Cascade polymer bound chelating compounds, their chelates and conjugates, processes for their production, and pharmaceutical agents containing them

ABSTRACT

Cascade polymers, containing complex-forming ligands, optionally at least five ions of an element of atomic numbers 21-29, 39, 42, 44 or 57-83, as well as, if desired, cations of inorganic and/or organic bases, amino acids or amino acid amides, are valuable complexing compounds and complexes for diagnostics and therapy.

BACKGROUND OF THE INVENTION

The invention relates to novel cascade polymer complexing compounds andcomplexes, agents containing these compounds, the use of the complexesin diagnostics and therapy, as well as processes for the production ofthese compounds and agents.

"Magnevist" (GdDTPA/dimeglumine) is the first recorded contrast mediumfor nuclear spin tomography (MRI =magnetic resonance imaging). It isparticularly well suited for the diagnosis of pathological areas (e.g.,inflammations, tumors, etc.). The compound is eliminated, uponintravenous injection, by way of the kidneys; extrarenal elimination ispractically not at all observed.

One disadvantage of "Magnevist" resides in that it is distributed afterintravenous administration uniformly between the vasal and interstitialspaces. Accordingly, contrasting of the vessels with respect to thesurrounding interstitial space is impossible with the use of"Magnevist".

Especially for the imaging of vessels, a contrast medium would bedesirable which is distributed exclusively in the vasal space (vascularspace). Such a blood pool agent is to make it possible, with the aid ofnuclear spin tomography, to demarcate tissue with good circulation fromtissue with poor circulation, and thus to diagnose an ischemia. Alsoinfarcted tissue could be distinguished, on account of its anemia, fromsurrounding healthy or ischemic tissue with the use of a vasal contrastmedium. This is of special importance in case the objective is, forexample, to distinguish a cardiac infarction from an ischemia.

Heretofore, most of those patients suspected of harboring acardiovascular disease (this disease being the most frequent cause ofdeath in Western industrial countries) had to undergo invasivediagnostic tests. In angiography, X-ray diagnostics is presently used,above all, with the aid of iodine-containing contrast media. These testsare burdened by various drawbacks: they bring the risk of radiationstress, as well as discomfort and strain stemming, above all, from thefact that the iodine-containing contrast media must be utilized in avery much higher concentration as compared with NMR contrast media.

Therefore, there is a need for NMR contrast media which can mark thevasal space (blood pool agent). These compounds are to be distinguishedby good compatibility and by high efficacy (great increase in signalintensity during MRI).

The premise of solving at least part of these problems by the use ofcomplexing agents bound to macro- or biomolecules has thus far beensuccessful to only a very limited extent.

Thus, for example, the number of paramagnetic centers in the complexesdescribed in European Patent Applications No. 88,695 and No. 150,884 isinadequate for satisfactory imaging.

When increasing the number of required metal ions by repeatedintroduction of complexing units into a macromolecule, the result is anintolerable impairment of the affinity and/or specificity of thismacromolecule [J. Nucl. Med. 24:1158 (1983)].

Macromolecules are generally suited as contrast media for angiography.Albumin-GdDTPA (Radiology 1987; 162:205), for example, shows, however,an accumulation in liver tissue to an extent of almost 30% of the dose24 hours after intravenous injection in rats. Besides, only 20% of thedose is eliminated within 24 hours.

The macromolecule polylysine-GdDTPA (European Patent Application,Publication No. 0,233,619) likewise proved to be suitable as a bloodpool agent. However, this compound, on account of its production,consists of a mixture of molecules of various sizes. In eliminationtests on rats, it could be demonstrated that this macromolecule iseliminated unchanged by glomerular filtration via the kidneys. Due toits synthesis, however, polylysine-GdDTPA can also containmacromolecules which are so large that they cannot pass through therenal capillaries during glomerular filtration and therefore remain inthe body.

Macromolecular contrast media based on carbohydrates, for exampledextran, have also been described (European Patent Application,Publication No. 0,326,226). The disadvantage of these compounds residesin that they carry normally only 4.6% of the signal-intensifyingparamagnetic cation.

SUMMARY OF THE INVENTION

An object, therefore, resides in making available novel diagnostic aids,above all for the recognition and localization of vascular diseases,which aids do not exhibit the aforedescribed disadvantages. This andother objects have been attained by the present invention.

It has been found that complexes comprising nitrogen-containing cascadepolymers provided with complexing ligands, ions of an element of atomicnumbers 21-29, 39, 42, 44 or 57-83, as well as optionally cations ofinorganic and/or organic bases, amino acids or amino acid amides, aresurprisingly excellently suitable for the production of NMR and X-raydiagnostic media without exhibiting the afore-mentioned drawbacks.

The polymers according to this invention can be described by generalFormula I ##STR1## wherein A means a nitrogen-containing cascade nucleusof basis multiplicity b,

S means a reproduction unit,

N means a nitrogen atom,

Z¹ and Z², for the first to penultimate generation in each case are##STR2## but, for the last generation, Z¹ means a hydrogen atom, a C₁-C₁₀ -alkyl, C₂ -C₁₀ -acyl (e.g., alkanoyl) or C₁ -C₁₀ -alkylsulfonylresidue, each optionally containing 1-3 carboxy, 1-3 sulfonic acid, 1-5hydroxy groups and/or 1-3 oxygen atoms (e.g., oxa(--O--) atoms), or itmeans the residue of a complexing agent or complex K, and

Z² means, to an extent of 96-100%, the residue of a complexing agent orcomplex K and, to an extent of 4-0%, V' wherein V' is the residue Vexhibiting at the end a functional group or, linked via this functionalgroup, a bio- or macromolecule, V meaning a straight-chain, branched,saturated or unsaturated C₁ -C₂₀ -alkylene group which optionallycontains imino, phenylene, phenylenoxy, phenylenimino, amide, hydrazide,ureido, thioureido, carbonyl, ester group(s), oxygen, sulfur and/ornitrogen atom(s) and is optionally substituted by hydroxy, mercapto,imino, epoxy, oxo, thioxo, and/or amino group(s),

b means the numbers 1 through 50, and

s means the numbers 1 to 3, wherein the reproduction units S can bedifferent from generation to generation. Also the complex (forming)residues optionally standing for Z¹ and Z.sup. 2 need not be identical.A "generation" is represented by each S group in a chain of S groups.

Examples of alkyl, acyl and alkylsulfonyl residues standing for Z¹ thatcan be cited are: --CH₂ COOH; --(CH₂)₂ COOH; --CH(COOH)CH₂ COOH; --CH₂--CH(COOH)CH₂ OH; --CH₂ SO₃ H; --(CH₂)₂ SO₃ H; --COCH₃ ; --COCH₂ OH;--COCHOHCH₂ OH; --COCH₂ O--CH₂ COOH; --CO(CHOH)₄ CH₂ OH; --COCH₂ COOH:--CO(CH₂)₂ COOH; --CO(CH₂)₃ COOH; --CO(CH₂)₄ COOH; --COCHOHCOOH:--CO(CHOH)₂ COOH; --COCH₂ CHOHCH₂ COOH; --SO₂ CH₂ COOH; --SO₂ (CH₂)₂COOH; --SO₂ CH₃.

Suitable as the cascade nucleus A are:

a nitrogen atom, ##STR3## wherein R² l , R³ and R⁴ mean in each caseindependently of one another, a covalent bond or --(CH₂)_(k) --(C₆H₄)_(r) --(CH₂)₁ --N<

g means the number 2, 3, 4 or 5,

t means the number 1, 2 , 3 , 4 , 5, 6, 7 or 8,

l means the number 0, 1, 2, 3, 4 or 5,

r means the number 0 or 1,

n means the number 0, 1, 2, 3 or 4,

m means the number 0, 1, 2, 3 or 4,

k means the number 1, 2, 3, 4 or 5,

a means the number 2 , 3, 4 or 5,

W means CH, CH₂, NH or a nitrogen atom,

C₁ means (CH₂)_(k) --N<,

C₂, C₃, C₄ and C₅ mean, in each case independently, a hydrogen atom or(CH₂)_(f) --N<,

f means the number 1, 2, 3, 4 or 5,

j means the number 6, 7 or 8,

Y¹ and Y² mean, in each case independently of each other, a hydrogenatom CH₂ --CH(OH)--CH₂ N< or (CH₂)_(n) --N<, and

Y³ is a nitrogen atom, O--CH₂ --CH(OH)--CH₂ N< or O--(CH₂)₉ --N<,

a and g are as defined above,

means a single or double bond, with the proviso that, if Y³ is anitrogen atom, Y¹ and Y² mean hydrogen. C₆ H₄ is phenylene.

The simplest case of a cascade nucleus is represented by the nitrogenatom, the three bonds of which (basis multiplicity b =3) are occupied ina first "inner layer" (generation 1) by three reproduction units S eachof which carries 1 to 3 terminal NH2 groups (s=1-3) (or, alternatively,the three hydrogen atoms of the basic cascade starter ammonia have beensubstituted by three units S). If the reproduction unit S contains, forexample, an NH₂ group (s=1), then the reproduction multiplicity of thisgeneration is 2 s=2. The second layer (generation 2) of reproductionunits S introduced in a subsequent reaction sequence (occupying, in theabove-mentioned example with A=nitrogen atom and s=1, six bonds) neednot be identical with the reproduction units S of the first generation.After preferably maximally 10, most preferably 2-6 generations, theterminal nitrogen atoms of the outermost layer are substituted asindicated above for Z¹ and Z² of the final generation.

Further preferred cascade starters A(H)_(b) that can be listed are,inter alia:

tris(aminoethyl)amine (b=6);

tris(aminopropyl)amine (b=6);

diethylenetriamine (b=5);

triethylenetetramine (b=6);

tetraethylenepentamine (b=7);

H₂ N--CH₂ --C₆ H₄ --CH₂ --NH--CH₂ --C₆ H₄ --NH₂ (b=5);

1,3,5-tris(aminomethyl)benzene (b=6);

2,4,6-tris(aminomethyl)pyridine (b=6);

1,4,7-triazacyclononane (b=3);

1,4,7,10-tetraazacyclododecane (b=4);

1,4,7,10,13-pentaazacyclopentadecane (b=5);

1,4,8,11-tetraazacyclotetradecane (b=4);

1,4,7,10,13,16,19,22,25,28-decaazacyclotriacontane (b=10);

6,6',6",6"',6"",6""'-hexaamino-6,6',6",6"',6"",6""'-hexadeoxy-α-cyclodextrin (b=12); 6,6',6",6"',6"",6""',6"""-heptaamino-6,6',6",6"',6"",6""',6"""-heptadeoxy-β-cyclodextrin(b=14);

6,6',6",6"',6"",6""'-hexa-(1-amino-2-hydroxypropyl)-α-cyclodextrinhexaether (b=12);

2,2',2",2"',2"",2""',6,6',6",6"',6"",6""'-dodeca-(1-amino-2-hydroxypropyl)-α-cyclodextrindodecaether (b=24).

Thus, b generally is the number of cascadable (reacting) nitrogenvalences (bonds) in an A group, e.g., corresponding to the number of Hatoms bonded to N atoms.

The reproduction unit S typically has the formula ##STR4## wherein a isa number 2, 3, 4 or 5,

α and β in each case mean a hydrogen atom or (CH₂)_(o),

γ means (CH₂)_(f),

k is 1, 2, 3, 4 or 5,

l is 0, 1, 2, 3, 4 or 5,

o is 0, 1, 2, 3, 4 or 5,

f means the number 1, 2, 3, 4 or 5, and

r means the number 0 or 1,

with the proviso that o and l are not both zero at the same time.

Preferred reproduction units S are ##STR5##

Suitable complex (forming) residues K are described by general Formula IA, I B and I C: ##STR6## wherein n and m in each case independently meanthe number 0, 1, 2, 3 or 4, n and m adding up to no more than 4,

k means the number 1, 2, 3, 4 or 5,

l means the number 0, 1, 2, 3, 4 or 5,

q means the number 0, 1 or 2,

U is CH₂ X or V,

X means in each case independently the residue --COOH or V' wherein, ifthe molecule contains V', at least 0.1% of the substituents X stand forV',

B, D and E, being identical or different, mean in each case the group--(CH₂)_(a) with a meaning the number 2, 3, 4 or 5,

R¹ means V or a hydrogen atom,

V and V' are as defined above,

with the proviso that R¹ means V only if U means CH₂ X at the same time,and that U means V only if R¹ means a hydrogen atom at the same time, aswell as with the proviso that, if desired, a portion of the COOH groupsis present as ester and/or amide.

Examples that can be cited for the complex forming residues K are thoseof ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,trans-1,2-cyclohexanediaminetetra-acetic acid,1,4,7,10-tetraazacyclododecanetetraacetic acid,1,4,7-triazacyclononanetriacetic acid,1,4,8,11-tetraazatetradecanetetraacetic acid,1,5,9-triazacyclododecanetriacetic acid,1,4,7,10-tetraazacyclododecanetriacetic acid and3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-trienetriaceticacid which are linked via (in each case contained in K) a carbonyl group(I A; I B and I C, contained in V, e.g., where U is V if R¹ means ahydrogen atom at the same time) or via a carbon atom (contained in V,see definition for U and R¹ as per above, e.g., in I B and I C where R¹is V if U stands for CH₂ X at the same time) to respectively oneterminal --NH₂ group of the final generation of the cascade polymer. Ifdesired, a portion of the carboxylic acids can be present as convertedto ester and/or amide groups.

As Z² of the last generation, V' can also be present up to a proportionof 4%.

Suitable complexes for Z¹ and Z² correspond to the foregoing complexing(chelating) agents as bonded (chelated) to central metal ions.

If the medium of this invention is intended for use in NMR diagnostics,then the central ion of the complex salt must be paramagnetic. Theseare, in particular, the di- and trivalent ions of the elements of atomicnumbers 21-29, 42, 44 and 58-70. Suitable ions are, for example, thechromium(III), manganese(II), iron(II), cobalt(II), nickel (II),copper(II), praseodymium(III), neodymium(III), samarium(III) andytterbium(III) ions. On account of their very strong magnetic moment,gadolinium(III), terbium(III), dysprosium(III), holmium(III),erbium(III) and iron(III) ions are especially preferred.

In case the agent of this invention is meant for use in X-raydiagnostics, the central ion must be derived from an element of a higheratomic number in order to obtain adequate absorption of the X rays. Ithas been found that diagnostic aids are suitable for this purpose whichcontain a physiologically compatible complex salt with central ions ofelements of atomic numbers between 21-29, 39, 42, 44, 57-83; these are,for example, the lanthanum(III) ion and the above-mentioned ions of thelanthanide series.

The cascade polymer complexes according to this invention contain atleast five of the ions of an element of the aforementioned atomicnumbers.

The alkylene group standing for V as well as the hydrocarbyl groupstanding for R and R' (below) can be straight-chain, branched, cyclic,aliphatic, aromatic or arylaliphatic and can contain up to 20 carbonatoms. Straight-chain mono- to decamethylene groups as well as C₁ -C₄-alkylenephenyl groups are preferred. The following alkylene groups arecited as examples for explanatory purposes: ##STR7## wherein R⁺ andR^(y) stand for natural amino acid residues; --CH₂ --(OH)--CH₂--O--(CH₂)₂ --NHCS--: --CH₂ --CH(OH)--CH₂ --NHCS--; --CH₂ --CH(OH)--CH₂--O--(CH₂)₂ --O--(CH₂)₂ NHCS--: --CH₂ --CH(OH)--CH₂ --O--(CH₂)₂--NH--CO--CH₂ --; --CH₂ --CH(OH)--CH₂ --O--C₆ H₄ --NHCS--: --CH₂--CH(OH)--CH₂ --O--C₆ H₄ --NHCO--; --CH₂ --CH(OH)--CH₂ --O--CH₂ --C₆ H₄--NHCS--; --CH₂ --O--C₆ H₄ --CH₂ --; --CH₂ --CH(OH)--CH₂ --O--C₆ H₄--CH₂ --; --C(═NH)--O--C₆ H₄ --CH₂ --; --(CH₂)₄ --NH--CO--CH₂ --O--C₆ H₄--CH₂ --; --(CH₂)₄ --NH--CH₂ --CH(OH)--CH₂ --O--C₆ H₄ CH₂ --; --(CH₂)₃--O--C₆ H₄ --CH₂ --; --CH₂) --CO--NH--(CH₂)₃ --O--CH₂ --: --CH₂--CO--NH--NH--: --CH₂ --CONH--(CH₂)₂ --; --CH₂ --CO--NH--(CH₂)₁₀ --;--CH₂ --CONH--(CH₂)₂ --S--: --(CH₂)₄ --NH--CO--(CH₂)₈ --; --CH₂--CO--NH--(CH₂)₃ --NH--; --(CH₂)₃ --NH-- groups,

Preferred functional groups located at the end of the V" alkylene groupare, for example, the maleimidobenzoyl, 3-sulfomaleimidobenzoyl,4-(maleimidomethyl) cyclohexylcarbonyl, 4-[3-sulfo(maleimidomethyl)cyclohexyl]carbonyl, 4-(p-maleimidophenyl) butyryl,3-(2-pyridyldithio)propionyl, methacryloyl(pentamethylene)amido,bromoacetyl, iodoacetyl, 3-iodopropyl, 2-bromoethyl, 3-mercaptopropyl,2-mercaptoethyl, phenyleneisothiocyanate, 3-aminopropyl, benzyl ester,ethyl ester, tert-butyl ester, amino, C₁ -C₆ -alkylamino, aminocarbonyl,hydrazino, hydrazinocarbonyl, maleimido, methacrylamido,methacryloylhydrazinocarbonyl, maleimidamido-carbonyl, halogeno,mercapto, hydrazinotrimethylenehydrazinocarbonyl,aminodimethyleneamidocarbonyl, bromocarbonyl, phenylenediazonium,isothiocyanate, semicarbazide, thiosemicarbazide, isocyanate groups.Several selected groups will be set forth for explanatory purposes:##STR8## wherein R and R' are identical or different and mean in eachcase a hydrogen atom, a saturated or unsaturated C₁ -C₂₀ -hydrocarbylresidue optionally substituted by a phenyl group, or a phenyl group.

The residual acidic hydrogen atoms, i.e. those that have not beensubstituted by the central ion, can be replaced, if desired, entirely orpartially by cations of inorganic and/or organic bases or amino acids.The corresponding acid groups can also be converted entirely orpartially into esters or amides.

Suitable inorganic cations are, for example, the lithium ion, thepotassium ion, the calcium ion, the magnesium ion, and especially thesodium ion. Suitable cations of organic bases are, inter alia, those ofprimary, secondary or tertiary amines, e.g. ethanolamine,diethanolamine, morpholine, glucamine, N,N-dimethylglucamine and, inparticular, N-methylglucamine. Suitable cations of amino acids are, forexample, those of lysine, of arginine and of ornithine, as well as theamides of otherwise acidic or neutral amino acids.

Suitable esters are preferably those with a C₁ -C₆ -alkyl residue;examples that can be cited are the methyl, ethyl and tert-butylresidues.

In case the carboxylic acid groups are to be present at least in part asamides, then tertiary amides are preferred. Suitable residues aresaturated, unsaturated, straight- or branched-chain or cyclichydrocarbons of up to 5 carbon atoms optionally substituted by 1-3hydroxy or C₁ -C₄ -alkoxy groups. Examples that can be cited are themethyl, ethyl, 2-hydroxyethyl, 2-hydroxy-1-(hydroxymethyl)ethyl,1-(hydroxymethyl)ethyl, propyl, isopropenyl, 2-hydroxypropyl,3-hydroxypropyl, 2,3-dihydroxypropyl, butyl, isobutyl, isobutenyl,2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2-, 3- and4-hydroxy-2-methylbutyl, 2- and 3-hydroxyisobutyl,2,3,4-trihydroxybutyl, 1,2,4-trihydroxybutyl, pentyl, cyclopentyl and2-methoxyethyl groups. The amide residue can also be a heterocyclic 5-or 6-membered ring formed with inclusion of the amide nitrogen. Examplesin this connection are: the pyrrolidinyl, piperidyl, pyrazolidinyl,pyrrolinyl, pyrazolinyl, piperazinyl, morpholinyl, imidazolidinyl,oxazolidinyl, thiazolidinyl rings.

The compounds of this invention exhibit the desirable properties setforth hereinabove. They contain the large number of metal ions, requiredfor their use, bound in the complex in a stable fashion. They aredistributed (if V' does not contain a bio- or macromolecule) only in thevasal space and thus can map this space with the aid of nuclear spintomography.

The compatibility of the compounds according to this invention isimproved by at least a factor of 3 over "Magnevist" (LD₅₀ i.v. mice ofExample 8: 30 mmol/kg; "Magnevist": ≦10) .

The value of osmolality, responsible for side effects, such as pain,damage to the blood vessels and cardiovascular disturbances, is reducedas compared with "Magnevist" (Example 8: 0.46 [osmol/kg] as comparedwith "Magnevist" 1.96 [osmol/kg], 0.5 mol/l at 37° C.).

The value for the magnitude of relaxation, representing a measure forimaging in MRI, is surprisingly high; signal intensification could beincreased over "Magnevist", for example in case of the compound ofExample 8, fourfold.

As compared with the macromolecular contrast media based oncarbohydrates, e.g. dextran (European Patent Application, PublicationNo. 0,326,226) which carry--as mentioned--normally only 4.6% of thesignal-intensifying paramagnetic cation, the polymer complexes of thisinvention contain more than 15% of the paramagnetic cation. Accordingly,the macromolecules of this invention bring about, per molecule, a verymuch higher signal intensification which has the result, at the sametime, that the dose required for nuclear spin tomography is considerablysmaller as compared with that for macromolecular contrast media based oncarbohydrates.

It has been made possible with the polymer complexes according to thisinvention to construct and produce macromolecules in such a way thatthey exhibit a uniformly defined molecular weight. Such macromolecularcontrast media, exactly definable in their molecular size, have not beenaccessible heretofore. It is thus surprisingly possible for the firsttime to regulate the size of the macromolecules so that these are largeenough to be able to leave the vasal space only gradually but, at thesame time, small enough to still pass through the kidney capillarieswhich have a size of 300-800 Å. It has thus been accomplished for thefirst time to produce macromolecular contrast media tailored to thebody.

The complexes of this invention serve as contrast media for imaging thevessels by means of nuclear spin tomography. It is thus possible todifferentiate between ischemic tissue and normal tissue. However, alsoother damage to the blood-tissue barrier can be recognized by means ofthese compounds. In case of inflammations and tumors in the brain, theblood-brain barrier is damaged to such an extent that the contrastmedium can infiltrate the diseased tissue and thus the diseased tissuebecomes visible in nuclear spin tomography. On account of theimpermeability of the intact blood-brain barrier, even to small, buthydrophilic molecules, inflammations and tumors have also beenrecognizable even with the low-molecular compound "Magnevist". However,when using the complexes of this invention in these cases, the dosagecan be reduced sixteenfold, for two reasons: (1) they exhibit a signalintensification which is four times higher, and (2) they are distributedin a space that is four times smaller, namely only in the vasal space,i.e. in order to reach the same concentrations in the blood, one-fourthof the dose is sufficient.

Another advantage of the present invention resides in that complexeswith hydrophilic or lipophilic, macrocyclic or open-chain, low-molecularor high-molecular ligands have now become accessible. This affords thepossibility of controlling compatibility and pharmacokinetics of thesepolymer complexes by chemical substitution.

By the choice of suitable bio- or macromolecules (see further below) inV', polymer complexes according to this invention are obtained whichexhibit a surprisingly high tissue and organ specificity.

The cascade polymers according to this invention are produced byreacting compounds of general Formula I' ##STR9## wherein A means anitrogen-containing cascade nucleus of the basis multiplicity b,

S means a reproduction unit,

N means a nitrogen atom, Z^(1') and Z^(2') mean the first to penultimategeneration, in each case ##STR10## but, for the final generation, ineach ease mean a hydrogen atom, b means the numbers 1 through 50, and

s means the numbers 1 to 3,

wherein the reproduction units S need to be identical only for onegeneration--optionally after reaction of up to 4% of the terminal aminogroups with a C₄ -C₂₀ -alkylene chain that is substituted at the ends bycarboxyl and hydrazide (preferably in the blocked form)--with a complexor complexing compound K' of the general formulae ##STR11## wherein nand m in each case are the number 0, 1, 2, 3 or 4, n and m adding up tono more than 4,

k means the number 1, 2, 3, 4 or 5,

l means the number 0, 1, 2, 3, 4 or 5,

q means the number 0, 1 or 2,

U' means --CH₂ C*O--, CH₂ X' or V" wherein V" stands for astraight-chain, branched, saturated or unsaturated C₁ -C₂₀ -alkylenegroup which optionally contains imino, phenylene, phenylenoxy,phenylenimino, amide, hydrazide, ureido, thioureido, carbonyl, estergroup(s), oxygen, sulfur and/or nitrogen atom(s) and is optionallysubstituted by hydroxy, mercapto, imino, epoxy, oxo, thioxo and/or aminogroup(s), this alkylene group carrying a functional group at the end,

X' means in each case independently the residues --COOH, COOY or V"',

wherein

Y is an acid blocking group or a metal ion equivalent of an element ofatomic numbers 21-29, 39, 42, 44 or 57-83, and V"' means a substituentto be converted into V',

C*O stands for an activated carbonyl group,

B, D and E, being identical or different, mean in each case the group(CH₂)_(a) where a means the number 2, 3, 4 or 5,

R^(1') is V" or a hydrogen atom, with the proviso that R^(1') stands forV" only if U' means CH₂ X' at the same time, and that U' means --CH₂C*O-- or V" only if R^(1') means a hydrogen atom at the same time,

optionally splitting off any blocking groups present, reacting, ifdesired, the thus-obtained cascade polymers--insofar as K' means acomplexing compound --conventionally with at least one metal oxide ormetal salt of an element of atomic numbers 21-29, 39, 42, 44 or 57-83,and optionally converting them into the cascade polymers carrying thedesired macro- or biomolecule(s) by conversion of at least one of the--CO₂ H-- or V"' groups contained in K' into the desired alkylene groupV" exhibiting a functional group at the end and optionally by subsequentlinkage via this functional group and/or via the terminal-positionedhydrazide group that may be contained in Z² with a macro- or biomoleculeand/or by linkage to the biotin or avidin residue, wherein the indicatedreaction steps (except for the macro- or biomolecule linkage which cantake place only after generating the functional group) can be performedin any desired sequence, and Optionally substituting, subsequently, inthe thus obtained polymer complexes any still present acidic hydrogenatoms entirely or partially by cations of inorganic and/or organicbases, amino acids or amino acid amides or converting the correspondingacid groups entirely or partially into esters or amides.

Examples of all activated carbonyl group in the complexes and/orcomplex-forming compounds K' are anhydride, p-nitrophenyl ester and acidchloride.

The alkylation or acylation effected for the introduction of thecomplex-forming units is carried out with substrates containing thedesired substituent K (possibly bound to a leaving group), or from whichthe desired substituent, optionally after modification by secondaryreaction(s), is generated by the reaction. Examples that can be citedfor the first-mentioned substrates are halogenides, mesylates, tosylatesand anhydrides. Among the second group are, for example, oxiranes,thiiranes, aziranes, α,β-unsaturated carbonyl compounds or theirvinylogs, aldehydes, ketones, isothiocyanates and isocyanates.

Examples of secondary reactions that can be mentioned are estercleavages, hydrogenations, esterifications, oxidations, etherificationsand alkylations, performed in accordance with literature methods knownto those skilled in the art.

Selected examples of the residues V" contained in K' are listed asfollows: ##STR12##

An example that can be cited is the reaction of the monoanhydride N³-(2,6-dioxomorpholinoethyl)-N⁶-(ethoxycarbonylmethyl)-3,6-diazaoctanedioic acid with the respectivelydesired cascade polymers, containing terminal amino groups, in water orin mixtures of water with, for example, dioxane, THF, DMF, DMSO oracetonitrile at an alkaline pH, preferably 8-10, i.e. with the additionof bases, such as, for example, sodium hydroxide, potassium hydroxide ortriethylamine, at temperatures of 0°-50° C., preferably at roomtemperature. For a complete reaction, a two- to threefold excess ofmonoanhydride, for example, is preferably employed.

A further possibility that can be mentioned is the reaction ofsubstituents K' exhibiting terminal-positioned aldehyde groups with therespectively desired cascade polymers containing terminal amino groups,with subsequent reduction of the thus-formed Schiff bases analogously tomethods known from the literature (Synthesis 1975, 135). Thethus-generated secondary amines can be converted into tertiary amines,amides or thioamides by subsequent acylation or alkylation withα,β-unsaturated esters containing optionally 1-3 carboxy, 1-3 sulfonicacid, 1-5 hydroxy residues and/or 1-3 oxygen atoms, alkyl halogenides,anhydrides, acid halogenides, or complexes and/or complexing compoundsK'. As examples of reaction partners which substitute the secondaryamino hydrogen atoms the following can be cited: ##STR13##

The aldehydes required herein as educts can be prepared from thecorresponding vicinal diols by oxidation with, for example, sodiummetaperiodate in an aqueous or alcoholic solution analogously to methodsknown from the literature (e.g. "Makromol. Chem." 182:1641 [1981]).

By pursuing a suitable course of reaction, for example adjusting the pHvalue or addition of amines, concomitantly introduced ester groups can,if desired, be saponified and aminolyzed, respectively.

Purification of the resultant cascade polymers takes place preferably byultrafiltration with membranes of a suitable pore size (e.g. "Areicon")or gel filtration on, for example, suitable "Sephadex" gels.

Analogously, for example, complexing compounds or complexes, derivedfrom isothiocyanate, epoxide or α-halogenoacetyl, are made to reactunder pH control in an aqueous medium with the desired cascade polymeramines.

The compounds I' required as the educts are known (for example, EuropeanPatent Applications, Publication Nos. 0,154,788 and 0,331,616, GermanPatent Application P 38 25 040.3) or they can be prepared from thecorresponding polyamines (wherein any present functional groups areoptionally blocked) by alkylation with an ester of general Formula II

    HalCH.sub.2 COOY'                                          (II)

wherein Hal means chlorine, bromine or iodine and Y' means a hydrogenatom, an alkali metal or an acid blocking group Y.

The reaction takes place in polar aprotic solvents, such as, forexample, dimethylformamide, dimethyl sulfoxide, acetonitrile, aqueoustetrahydrofuran or hexamethylphosphoric triamide in the presence of anacid captor, such as, for example, a tertiary amine (e.g. triethylamine,trimethylamine, N,N-dimethylaminopyridine,1,5-diazabicycl[4.3.0]nonene-5 (DBN), 1,5-diazabicycl[5.4.0]undecene-5(DBU), alkali, alkaline earth carbonate, bicarbonate or hydroxide (e.g.sodium, magnesium, calcium, barium, potassium carbonate, hydroxide andbicarbonate) at temperatures of between -10° C. and 120° C., preferablybetween 0° C. and 50° C.

Suitable acid blocking groups Y are lower alkyl, aryl and aralkylgroups, for example the methyl, ethyl, propyl, butyl, phenyl, benzyl,diphenylmethyl, triphenylmethyl, bis(p-nitrophenyl)-methyl groups, aswell as trialkylsilyl groups.

The splitting off of the blocking groups Y which may be desirable takesplace according to the methods known to one skilled in the art, forexample by hydrolysis, hydrogenolysis, alkaline saponification of theesters with an alkali in an aqueous-alcoholic solution at temperaturesof 0° C. to 50° C. or, in case of tert-butyl esters, with the aid oftrifluoroacetic acid.

Production of the derivatives with an activated carbonyl group C*O, I'Aor I'B and I'C wherein U' means CH₂ C*O (e.g. mixed anhydride,N-hydroxysuccinimide ester, acylimidazoles, trimethylsilyl ester) takesplace according to methods known from the literature [Houben-Weyl,"Methoden der organischen Chemie" [Methods of Organic Chemistry], GeorgThieme publishers, Stuttgart, vol. E 5 (1985), 633; Org. React. 12:157(1962)]or will be described in the experimental portion.

Preparation of the cyclic polyamines needed as educts for I'B and I'Ctakes place by cyclization of two reactants of which--in case of thesynthesis of I'B with=R^(1') =V" --one is V"'-substituted, or (in caseof the synthesis of I'C) one contains the desired 6-membered ring of thefinal product or a precursor to be converted into this ring.

The cyclization is carried out according to methods known from theliterature [for example, Org. Synth. 58:86 (1978), Macrocyclic PolyetherSyntheses, Springer publishers, Berlin, Heidelberg, New York (1982),Coord. Chem. Rev. 3:3 (1968), Ann. Chem. 1976:916, J. Org. Chem. 49:110(1984)]; one of the two reactants carries two leaving groups at thechain end, the other carries two nitrogen atoms which displace theseleaving groups in nucleophilic fashion. An example that can be cited isthe reaction of terminal-positioned dichloro, dibromo, dimesyloxy,ditosyloxy or dialkoxycarbonyl alkylene compounds, containing, ifdesired, the-substituent V"'and optionally one to five nitrogen atom(s),with terminal-positioned polyazaalkylene compounds optionally containingone to five additional nitrogen atom(s) in the alkylene chain. Thesubstituent V"'can, instead, also be contained in the second reactant,i.e. the one having the terminal-positioned nucleophilic nitrogen atoms.The nitrogen atoms are blocked, if necessary, for example as tosylatesor trifluoroacetates, and they are liberated according to methods knownin the literature prior to the subsequent alkylation reaction (thetosylates, for example, with mineral acids, alkali metals in liquidammonia, hydrobromic acid and phenol, "RedAl", lithium aluminum hydride,sodium amalgam, compare, for example, Liebigs Ann. Chem. 1977:1344,Tetrahedron Letters 1976:3477; the trifluoroacetates, for example, withmineral acids or ammonia in methanol, compare, for example, TetrahedronLetters 1967:289).

For preparing macrocycles differently substituted on the nitrogen atoms(hydrogen or the group CH₂ COOY), these atoms can be provided in theeducts with differing blocking groups, for example with tosylate andbenzyl groups. The latter are then likewise removed according to methodsknown in the literature (preferably by hydrogenation, e.g. EP PatentApplication 232,751).

In case diesters are used in the cyclization reaction, the resultantdiketo compounds must be reduced by methods known to a person skilled inthe art, for example with diborane.

It is also possible to cyclize correspondingly substitutedterminal-positioned bisaldehydes with the respectively desiredterminal-positioned bisamines; the reduction of the thus-obtained Schiffbases takes place according to methods known in the literature, forexample by catalytic hydrogenation [Helv. Chim. Acta 61:1376 (1978)].

The amines required to serve as starting materials for the cyclizationare prepared in analogy to methods known from the literature.

Starting with an N-blocked amino acid, reaction with a partially blockeddiamine (e.g. according to the carbodiimide method), splitting off ofthe blocking groups, and diborane reduction yield a triamine.

Reaction of a diamine obtained from amino acids [Eur. J. Med.Chem.-Chim. Ther. 21:333 (1986)] with twice the molar amount of anN-protected ω-amino acid yields a tetramine after appropriate working upprocedure.

The desired diamines can also be prepared by Gabriel reaction from, forexample, the corresponding tosylates or halogenides [compare, forexample, Inorg. Chem. 25:4781 (1986)].

In both cases, the number of carbon atoms between the N atoms can bedetermined by the type of diamines or amino acids utilized as couplingpartners.

Conversion of a precursor of I'C, obtained by cyclizing, into thedesired complexing compound takes place according to methods known toone skilled in the art, for example deoxygenation of nitroxide [E.Klingsberg, The Chemistry of Heterocyclic Compounds, vol. 14, part 2,Interscience Publishers, New York, page 120, 1961) rings, conversions,and introduction of functional groups at the pyridine ring, e.g.liberation of phenolic hydroxy groups [J. Org. Chem. 53:5 (1988)],introduction of halogen substituents [E. Klingsberg, The Chemistry ofHeterocyclic Compounds, vol. 14, part 2, Interscience Publishers, NewYork, page 341, 1961; Houben-Weyl, "Methoden der organischen Chemic",vol. V/3, 651 (1962)].

Functionalization of 4-halopyridine derivatives (e.g. azide exchange) inthe phase transfer process with the use of 18-crown-6 ortetrabutylammonium halogenide as the catalyst has been described in"Phase Transfer Reactions" (Fluka Compendium vol. 2, Walter E. Keller,Georg Thieme publishers, Stuttgart, New York). A thus-obtained azidegroup can be converted into an amino function in accordance with methodsknown to one skilled in the art (e.g. catalytic hydrogenation,Houben-Weyl, "Methoden der organischen Chemie", vol. 11/1, p. 539; orreaction with Raney nickel/hydrazine, German Patent Application3,150,917). This amino function can be converted into an isothiocyanategroup according to methods known from the literature (for example withthiophosgene in a two-phase system, S. Scharma, Synthesis 1978:803; D.K. Johnson, J. Med. Chem. 1989, vol. 32, 236).

By reacting an amino function with a haloacetic acid halogenide, anα-halogenoacetamide group can be generated (JACS 1969, vol. 90, 4508;Chem. Pharm. Bull. 29 (1) : 128, 1981) which is suitable for coupling tobio- or macromolecules or cascade polymers in the same way as, forexample, the isothiocyanate group.

As a substituent V"' which can be converted into V, or into thesubstituent V" exhibiting at the end a functional group suitable forlinking to a macro- or biomolecule or to a cascade polymer, suitableare, inter alia, hydroxy and nitrobenzyl, hydroxy and carboxyalkyl, aswell as thioalkyl residues of up to 20 carbon atoms. They are converted,according to literature methods known to one skilled in the art [Chem.Pharm. Bull. 33:674 (1985), Compendium of Org. Synthesis, vol. 1-5,Wiley and Sons, Inc., Houben-Weyl, "Methoden der organischen Chemie",vol. VIII, Georg Thieme publishers, Stuttgart, J. Biochem. 92:1413(1982)], into the desired substituents (e.g. with the amino, hydrazino,hydrazinocarbonyl, epoxide, anhydride, methacryloylhydrazinocarbonyl,maleimidamidocarbonyl, halo, halocarbonyl, mercapto, isothiocyanategroup as the functional group) where, in case of the nitrobenzylresidue, first a catalytic hydrogenation to the aminobenzyl derivativemust be performed (for example according to P. N. Rylander, CatalyticHydrogenation Over Platinum Metals, Academic Press, 1967).

Examples of the conversion of hydroxy or amino groups bound to aromaticor aliphatic residues are the reactions carried out with a substrate ofgeneral Formula III

    Nf--L--Fu                                                  (III)

wherein

Nf means a nucleofugal entity, such as, for example, Cl, Br, I, CH₃ C₆H₄ SO₃ or CF₃ SO₃,

L is an aliphatic, aromatic, arylaliphatic, branched, straight-chain orcyclic hydrocarbon residue of up to 20 carbon atoms, and

Fu is the desired, terminal-positioned functional group, optionally inthe blocked form (DOS 3,417,413),

performed in suitable solvents, such as tetrahdyrofuran, dimethoxyethaneor dimethyl sulfoxide, two-phase aqueous systems, such as, for example,water/dichloromethane, in the presence of an acid captor, such as, forexample, sodium hydroxide, sodium hydride or alkali or alkaline earthcarbonates, such as, for example, sodium magnesium, potassium, calciumcarbonate or poly-(4-vinylpyridine) "Reillex", at temperatures ofbetween 0° C. and the boiling point of the respective solvent, butpreferably between 20° C. and 60° C.

Examples of compounds according to general Formula III are: ##STR14##

Conversions of carboxy groups can be performed, for example, accordingto the carbodiimide method (Fieser, Reagents for Organic Synthesis 10,142), by way of a mixed anhydride [Org. Prep. Proc. Int. 7: 215 (1975)]or by way of an activated ester (Adv. Org. Chem., part B, 472).

Introduction of the optionally desired substituent V" at a nitrogen atomof the complexing compounds I'B and I'C (i.e., U'=V") can likewise beeffected according to the above-mentioned process, i.e. here, too, amacrocycle intermediate stage containing V"' is usually passed throughwhich is obtained by reaction of a polyaza macrocycle exhibiting onlyone free NH group. Examples in this connection are the reaction of, forexample, 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclocodecane with aprimary epoxide exhibiting a blocked amino group, subsequent liberationof the amino function of the resultant V"'-substituted macrocycle, andsubsequent conversion into a V"-substituted macrocycle (for example,conversion of the amino group into a functional group that can becoupled to the cascade polymer amine, such as, for example, theisothiocyanate or 2-halogenoacetamide group).

The cascade polymers carrying terminal amino groups, needed for couplingto the complexing compounds K (and/or also the correspondingmetal-containing complexes), are prepared according to methods known topersons skilled in the art by a cascade-type, generation-wiseintroduction of nitrogen atoms into a nitrogen-containing basismolecule. This yields a generation from at least two reaction steps.From each amino hydrogen atom of the cascade starter, up to three aminogroups are generated in this way by, for example, a Michael addition oraddition of a primary epoxide containing a suitable functional group,and subsequent conversion of the thus-introduced functional group.

An example that can be cited is the substitution of the six aminohydrogen atoms of the cascade starter tris(aminoethyl)amine by six --CH₂CH₂ --CONH--CH₂ CH₂ NH₂ units obtained by Michael addition with acrylicacid ester and subsequent aminolysis with ethylenediamine. Theaminolysis, preferably performed without solvents, is here conductedwith an up to 500-fold amine excess per ester grouping at temperaturesof 0° C. to about 130° C.

As an example of an epoxide addition, the reaction can be cited of6,6',6",6'",6"",6""'-hexaamino-6,6',6",6'",6"",6""'-hexadeoxy-α-cyclodextrinwith 1,3-(N,N'-tetrabenzyl)diamino-2-(oxiranylmethoxy)propane andsubsequent liberation of the amino functions by catalytic hydrogenationin accordance with methods known to one skilled in the art (see alsoabove).

A portion of the acid groups of the thus-obtained polymer compounds,introduced via the complex forming units K, can be furtherfunctionalized, if desired, according to processes known to a personskilled in the art, for example by converting into ester, amide,hydrazide, maleimido or other groups suitable for coupling to bio- ormacromolecules.

The thus-obtained complexing ligands (as well as the complexes) can alsobe linked to bio- or macromolecules from which it is known that they areparticularly accumulated in the organ or organ part to be examined. Suchmolecules are, for example, enzymes, hormones, polysaccharides, such asdextrans or starches, porphyrins, bleomycins, insulin, prostaglandins,steroid hormones, amino sugars, amino acids, peptides such aspolylysine, proteins (such as, for example, immunoglobulins, monoclonalantibodies, lectins), lipids (also in the form of liposomes), andnucleotides of the DNA or RNA type. Especially to be emphasized areconjugates with albumins, such as human serum albumin, antibodies, e.g.monoclonal antibodies specific for tumor-associated antigens, orantimyosin. Instead of biological macromolecules, it is also possible tolink suitable synthetic polymers, such as polyethylenimines, polyamides,polyureas, polyethers, such as polyethylene glycols, and polythioureas.The pharmaceutical agents formed therefrom are suitabe, for example, foruse in tumor and infarction diagnostics, as well as tumor therapy.Monoclonal antibodies (e.g. Nature 256:495, 1975) have the advantagesover polyclonal antibodies that they are specific for an antigendeterminant, that they possess definite binding affinity, that they arehomogeneous (thus substantially simplifying their production in pureform), and that they can be manufactured in large amounts in cellcultures. Suitable are, for example, for tumor imaging, monoclonalantibodies and/or their fragments Fab and F(ab')₂ which are specific,for example, for human tumors of the gastrointestinal tract, of thebreast, of the liver, of the bladder, of the gonads, and of melanomas[Cancer Treatment Repts. 68:317 (1984), Bio. Sci. 34:150 (1984)]or aredirected against carcinomembryonal antigen (CEA), human chorionicgonadotropin (β-HCG), or other tumor-positioned antigens, such asglycoproteins [New Engl. J. Med. 298:1384 (1973), U.S. Pat. No.4,331,647]. Suitable are, inter alia, also antimyosin, anti-insulin andantifibrin antibodies (U.S. Pat. No. 4,036,945).

Colon carcinomas can be confirmed by NMR diagnosis with the aid ofconjugates complexed with gadolinium(III) ions, using the antibody 17-1A(Centocor, USA).

For liver examinations and tumor diagnostics, respectively, conjugatesor inclusion compounds are suitable, for example, with liposomesutilized, for instance, as unilamellar or multilamellarphosphatidylcholine cholesterol vesicles.

Heretofore, bonding of metals to the desired macro- or biomolecules hasbeen performed according to methods described, for example, in Rev.Roum. Morphol. Embryol. Physio., Physiologie 1981, 18:241, and in J.Pharm. Sci. 68:79 (1979), e.g. by reaction of the nucleophilic group ofa macromolecule, such as the amino, phenol, sulfhydryl, aldehyde orimidazole group, with an activated derivative of the polymer complex orligand. Examples of activated derivatives are anhydrides, acidchlorides, mixed anhydrides (see, for example, G. E. Krejcarek and K. L.Tucker, Biochem., Biophys. Res. Commun. 1977, 581), activated esters,nitrenes or isothiocyanates. Conversely, it is also possible to react anactivated macromolecule with the polymer complex or ligand. Forconjugation with proteins, also suitable are, for example, substituentsof the structure C₆ H₂ N₂ ⁺, C₆ H₄ NHCOCH₂ Br, C₆ H₄ NCS or C₆ H₄ OCH₂COBr.

However, this type of linkage is burdened by the drawback of lack incomplex stability of the conjugates and/or lack of specificity (forinstance, Diagnostic Imaging 84:56; Science 220:613, 1983; Cancer DrugDelivery 1:125, 1984). The conjugate formation according to the presentinvention takes place, in contrast thereto, via the functional groupspresent in V'. It is possible herein to bind up to more than one-hundredmetal ions via one binding site in the macromolecule.

In case of the antibody conjugates, binding of the antibody to thecomplex or ligand must not lead to loss or reduction of binding affinityand binding specificity of the antibody to the antigen. This can beaccomplished either by binding to the carbohydrate portion in the Fcpart of the glycoprotein and/or in the Fab or F(ab')₂ fragments, or bybinding to sulfur atoms of the antibody and/or antibody fragments.

In the first instance, an oxidative cleavage of sugar units must firstbe performed for the generation of formyl groups capable of coupling.This oxidation can be carried out by chemical methods with oxidizingagents such as, for example, periodic acid, sodium metaperiodate, orpotassium metaperiodate in accordance with methods known from theliterature (e.g., J. Histochem. and Cytochem. 22:1084, 1974) in anaqueous solution in concentrations of 1-100 mg/ml, preferably 1-20mg/ml, and with a concentration of the oxidizing agent of between 0.001to 10 millimoles, preferably 1 to 10 millimoles, in a pH range of about4 to 8 at a temperature of between 0° and 37° C. and with a reactionperiod of between-15 minutes and 24 hours. The oxidation can also beperformed by enzymatic methods, for example with the aid of galactoseoxidase in an enzyme concentration of 10-100 units/ml, a substrateconcentration of 1-20 mg/ml, at a pH of 5 to 8, a reaction period of 1-8hours, and a temperature of between 20° and 40° C. (for example, J.Biol. Chem. 234:445, 1959).

Complexes or ligands with suitable functional groups, such as, forexample hydrazine, hydrazide, hydroxylamine, phenylhydrazine,semicarbazide and thiosemicarbazide, are bound to the aldehydesgenerated by oxidation; this is done by reacting between 0° and 37° C.with a reaction period of 1-65 hours, a pH of between about 5.5 and 8,an antibody concentration of 0.5-20 mg/ml, and a molar ratio of thecomplexing compound to the antibody aldehyde of 1:1 to 1000:1. Thesubsequent stabilization of the conjugate takes place by reduction ofthe double bond, for example with sodium borohydride or sodiumcyanoborohydride; the reducing agent is utilized herein with a 10- to100-fold excess (e.g., J. Biol. Chem. 254:4359, 1979).

The second possibility of forming antibody conjugates starts with agentle reduction of the disulfide bridges of the immunoglobulinmolecule; in this process, the more sensitive disulfide bridges betweenthe H chains of the antibody molecule are cleaved whereas the S-S bondsof the antigen-binding region remain intact so that there is practicallyno reduction in binding affinity and specificity of the antibody(Biochem. 18:2226, 1979; Handbook of Experimental Immunology, vol. 1,2nd ed., Blackwell Scientific Publications, London 1973, chapter 10).These free sulfhydryl groups of the inter-H-chain regions are thenreacted with suitable functional groups of complexing compounds or metalcomplexes at 0°-37° C., a pH of about 4-7, and a reaction period of 3-72hours with the formation of a covalent bond which does not affect theantigen binding region of the antibody. Suitable reactive groups are,for example: haloalkyl, haloacetyl, p-mercuribenzoate, isothiocyanate,thiol, epoxy groups, as well as groups to be subjected to a Michaeladdition reaction, such as, for example, maleinimides, methacrylo groups(e.g. J. Amer. Chem. Soc. 101:3097, 1979).

Additionally, for linking the antibody fragments with the polymercomplexes or with the ligands, there is a number of suitablebifunctional "linkers" which are frequently also obtainable commercially(see, for example, Pierce, Handbook and General Catalogue 1986) whichare reactive with respect to the SH groups of the fragments as well aswith respect to the amino or hydrazino groups of the polymers.

Examples that can be cited are:

m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),

m-maleimidobenzoyl-N-sulfosuccinimide ester (Sulfo-MBS),

N-succinimidyl-[4-(iodoacetyl)amino]benzoic acid ester (SIAB),

succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid ester(SMCC),

succinimidyl-4-(p-maleimidophenyl)butyric acid ester (SMPB),

N-succinimidyl-3-(2-pyridyldithio)propionic acid ester (SDPD),

4-[3-(2,5-dioxo-3-pyrrolinyl)propionyloxy]-3-oxo-2,5-diphenyl-2,3-dihydrothiophene-1,1-dioxide,

acetylalanylleucylalanylaminobenzyl,

acetamido-p-thioureidobenzyl.

It is also possible to utilize bonds not of the covalent type forcoupling purposes wherein ionic as well as van der Waals and hydrogenbridge bonds can contribute toward the linkage in varying proportionsand strengths (key and lock principle) (for example, avidin-biotin,antibody-antigen). Also inclusion compounds (host-guest) of relativelysmall complexes in relatively large cavities in the macromolecule arepossible.

The coupling principle resides in first producing a bifunctionalmacromolecule by either fusing an antibody hybridoma directed against atumor antigen with a second antibody hybridoma directed against acomplex according to this invention, or linking the two antibodieschemically via a linker (e.g. in the way set forth in J. Amer. Chem.Soc. 101:3097, 1979) or binding the antibody directed against the tumorantigen to avidin (or biotin, respectively), optionally via a linker [D.J. Hnatowich et al., J. Nucl. Med. 28:1294 (1987)]. In place of theantibodies, it is also possible to employ their corresponding F(ab) orF(ab')₂ fragments. For pharmaceutical usage, first the bifunctionalmacromolecule is injected which is accumulated at the target site, andthen, at a time interval, the complex compound of this invention isinjected [optionally bound to biotin (or avidin)] which is coupled on atthe target site in vivo and there can deploy its diagnostic ortherapeutic activity. Moreover, other coupling methods can likewise beutilized, such as, for example, "reversible radiolabeling" described inProtein Tailoring Food Med. Uses [Am. Chem. Soc. Symp. 349 (1985)].

A particularly simple method for the production of antibody conjugatesor antibody fragment conjugates is available in the form of theso-called solid phase coupling procedure: The antibody is coupled to astationary phase (e.g. an ion exchanger) located, for example, in aglass column. By successive flushing of the column with a solutionsuitable for generation of aldehyde groups, washing, rinsing with asolution of the functionalized complex (or ligand), washing (in case theligand is used, rinsing is furthermore performed with a solutioncontaining the metal salt, followed by another rinsing step) and,finally, elution of the conjugate, very high conjugate yields areobtained.

This procedure permits the automatic and continuous production of anydesired quantities of conjugates.

Also other coupling steps can be performed in this way.

Thus, for example, fragment conjugates can be prepared by the sequenceof papain reduction/bifunctional linker/functionalized complex orligand.

The thus-formed compounds are subsequently purified preferably bychromatography by way of ion exchangers on a fast protein liquidchromatography unit.

The metal complexes of this invention are produced as disclosed inGerman Laid-Open Application 3,401,052 by dissolving or suspending themetal oxide or a metallic salt (e.g. the nitrate, acetate, carbonate,chloride or sulfate) of the element of atomic numbers 21-29, 42, 44,57-83 in water and/or in a lower alcohol (such as methanol, ethanol orisopropanol), and reacting with a solution or suspension of theequivalent amount of the complexing ligand and subsequently, if desired,substituting any acidic hydrogen atoms present in the acid or phenolgroups by cations of inorganic and/or organic bases or amino acids.

Introduction of the desired metal ions can take place at the stage ofthe complexing compounds I'A, I'B or I'C, i.e. prior to coupling to thecascade polymers, as well as after the coupling of the unmetalatedligands I'A, I'B or I'C.

Neutralization takes place herein with the aid of inorganic bases (e.g.hydroxides, carbonates or bicarbonates) of, for example, sodium,potassium, lithium, magnesium or calcium and/or organic bases, such as,inter alia, primary, secondary and tertiary amines, e.g. ethanolamine,morpholine, glucamine, N-methyl- and N,N-dimethylglucamine, as well asbasic amino acids, such as, for example, lysine, arginine and ornithine,or of amides from originally neutral or acidic amino acids.

In order to prepare the neutral complex compounds, it is possible, forexample, to add to the acidic complex salts in an aqueous solution orsuspension such an amount of the desired bases that the neutral point isobtained. The resultant solution can then be concentrated to drynessunder vacuum. It is frequently advantageous to precipitate thethus-formed neutral salts by adding water-miscible solvents, e.g. loweralcohols (methanol, ethanol, isopropanol and others), lower ketones(acetone and others), polar ethers (tetrahydrofuran, dioxane,1,2-dimethoxyethane and others) and to obtain in this way crystallizedproducts which can be easily isolated and readily purified. It proved tobe especially-advantageous to add the desired base as early as duringthe complex formation to the reaction mixture and thereby to save aprocess step.

In case the acidic complex compounds contain several free acidic groups,it is frequently expedient to produce neutral mixed salts containinginorganic as well as organic cations as the counterions.

This can be done, for example, by reacting the complex forming ligand inan aqueous suspension or solution with the oxide or salt of the elementyielding the central ion, and with half the amount of an organic baserequired for neutralization; isolating the thus-formed complex salt;purifying same if desired; and then combining, for completeneutralization, with the needed amount of inorganic base. The sequenceof addition of the bases can also be reversed.

Another possibility of obtaining neutral complex compounds resides inconverting the remaining acid groups in the complex entirely orpartially into esters or amides, for example. This can be done bysubsequent reaction at the finished polymer complex (e.g. by exhaustivereaction of the free carboxy groups with dimethyl sulfate), as well asalso by the use of a suitably derivatized substrate for introducing thecomplexing units of general Formulae I'A, I'B and I'C (e.g.N3-(2,6-dioxomorpholinoethyl)-N⁶-(ethoxycarbonylmethyl)-3,6-diazaoctanedioic acid).

The conjugates of antibody and complex are dialyzed, prior to in vivouse, after incubation with a weak complexing agent, such as, forexample, sodium citrate, sodium ethylenediaminetetraacetic acid, inorder to remove weakly bound metal atoms.

The pharmaceutical agents of this invention are likewise produced in amanner known per se by suspending or dissolving the complex compounds ofthis invention--optionally combined with the additives customary ingalenic pharmacy--in an aqueous medium and then optionally sterilizingthe suspension or solution. Suitable additives are, for example,physiologically acceptable buffers (such as, for instance,tromethamine), additions of complexing agents (e.g.diethylenetriaminepentaacetic acid) or--if required--electrolytes, e.g.sodium chloride or--if necessary--antioxidants, such as ascorbic acid,for example.

If suspensions or solutions of the compounds of this invention in wateror physiological saline solution are desirable for enteraladministration or other purposes, they are mixed with one or several ofthe auxiliary agents (e.g. methylcellulose, lactose, mannitol) and/ortensides (e.g. lecithins, "Tween", "Myrj") and/or flavoring agents toimprove taste (e.g. ethereal oils), as customary in galenic pharmacy.

In principle, it is also possible to produce the pharmaceutical agentsof this invention without isolating the complex salts. In any event,special care must be taken to effect chelate formation so that the saltsand salt solutions according to this invention are practically devoid ofuncomplexed, toxically active metal ions.

This can be ensured, for example, with the aid of dye indicators, suchas xylenol orange, by control titrations during the manufacturingprocess. Therefore, the invention also concerns processes for theproduction of the complex compounds and their salts. A final safetymeasure resides in purifying the isolated complex salt.

The pharmaceutical agents of this invention preferably contain 1 μmol to1 mol/l of the complex salt and are normally made into doses in amountsof 0.0001-5 mmol/kg. They are intended for enteral andparenteral-administration. The complex compounds according to thisinvention are utilized

(1) for NMR and X-ray diagnostics in the form of their complexes withthe ions of the elements with atomic numbers 21-29, 39, 42, 44 and57-83;

(2) for radiodiagnostics and radiotherapy in the form of their complexeswith the radioisotopes of the elements with atomic numbers 27, 29, 31,32, 37-39. 43, 49, 62,-64, 70, 75 and 77.

The agents of this invention meet the variegated requirements for beingsuitable as contrast media for nuclear spin tomography. Thus, they areexcellently suited for improving the informative content of the imageobtained with the aid of the NMR tomograph upon oral or parenteraladministration, by increasing the signal intensity. Furthermore, theyexhibit the high efficacy necessary to introduce into the body a minimumamount of burdening foreign substances, and they show the goodcompatibility required for maintaining the noninvasive character of theexaminations.

The good water solubility and low osmolality of the compounds of thisinvention make it possible to prepare highly concentrated solutions,thus maintaining the volume load on the circulation within tolerablelimits and compensating for dilution by body fluid, i.e. NMR diagnosticaids must exhibit. 100-1,000 times the water solubility of agents forNMR spectroscopy. Furthermore, the agents of this invention exhibit notonly a high in vitro stability but also a surprisingly high stability invivo so that release or exchange of the ions--actually toxic--not boundin a covalent fashion in the complexes takes place only extremelygradually within the time period during which the novel contrast mediaare again completely eliminated.

In general, the agents of this invention are used, for NMR diagnosticaids, in doses amounting to 0.0001-5 mmol/kg, preferably 0.005-0.5mmol/kg. Details of use are discussed, for example, in H. J. Weinmann etal., Am. J. of Roentgenology 142:619 (1984).

Especially low doses (below 1 mg/kg body weight) of organ-specific NMRdiagnostic aids are usable, for example, for the detection of tumors andof cardiac infarction.

Furthermore, the complex compounds according to this invention can beemployed with advantage as susceptibility reagents and as shift reagentsfor in vivo NMR spectroscopy.

The agents of this invention, based on their favorable readioactiveproperties and good stability of the complex compounds containedtherein, are also suited as radiodiagnostic agents. Details of theirusage and dosage are described, for example, in "Radiotracers forMedical Applications", CRC Press, Boca Raton, Fla.

Another imaging method with radioisotopes is the positron emissiontomography, using positron-emitting isotopes, such as, for example, ⁴³Sc, ⁴⁴ Sc, ⁵² Fe, ⁵⁵ Co and ⁶⁸ Ga (Heiss, W. D.; Phelps, M. E.: PositronEmission Tomography of Brain, Springer publishers, Berlin, Heidelberg,New York 1983).

The compounds of this invention can also be utilized in radioimmuno- orradiation therapy. This process differs from the correspondingdiagnostics only in the quantity and type of isotope employed. Theobjective herein is the destruction of tumor cells by high-energyshortwave radiation with a minimum range. Suitable B-emitting ions are,for example, ⁴⁶ Sc, ⁴⁷ Sc, ⁴⁸ Sc, ⁷² Ga, ⁷³ Ga and ⁹⁰ Y. Suitableα-emitting ions exhibiting short half-life periods are, for example, ²¹¹Bi, ²¹² Bi, ²¹³ Bi and ²¹⁴ Bi, wherein ²¹²

Bi is preferred. A suitable ion emitting photons and electrons is ¹⁵⁸ Gdwhich can be obtained from ¹⁵⁷ Gd by neutron capture.

If the agent of this invention is intended for use in the version ofradiation therapy proposed by R. L. Mills et al. [Nature, vol. 336:787(1988)], then the central ion must be derived from a Mossbauer isotope,such as, for example, ⁵⁷ Fe or ¹⁵¹ Eu.

In the in vivo administration of the therapeutic agents according tothis invention, they can be given together with a suitable carrier, e.g.serum or physiological sodium chloride solution and together withanother protein, such as, for example, human serum albumin. The dosageherein is dependent on the type of cellular disorder, the metal ionused, and the type of imaging method.

The therapeutic media of this invention are, e.g., administeredparenterally, preferably intravenously.

Details of usage of radiotherapeutic agents.. are discussed, forexample, in R. W. Kozak et al., TIBTEC, October 1986, 262.

The agents of this invention are excellently suited as X-ray contrastmedia; in this connection, it is to be especially emphasized that theyreveal no indication of anaphylaxis-type reactions, known fromiodine-containing contrast media, in biochemical-pharmacologicalstudies. They are particularly valuable, on account of the favorableabsorption properties in regions of higher tube voltages, for digitalsubtraction techniques.

In general, the agents of this invention are utilized, foradministration as X-ray contrast media, analogously to, for example,meglumine diatrizoate, in doses amounting to 0.1-5 mmol/kg, preferably0.25-1 mmol/kg.

Details of utilization of X-ray contrast media are discussed, forexample, in Barke, "Rontgenkontrastmittel" [X-Ray Contrast Media], G.Thieme, Leipzig (1970) and P. Thurn, E. Bucheler, "Einfuhrung in dieRontgendiagnostik" [Introduction to X-Ray Diagnostics], G. Thieme,Stuttgart, New York (1977).

In summation, the synthesis has been accomplished of novel complexingcompounds, metal complexes and metal complex salts, opening up newpossibilities in diagnostic and therapeutic medicine. This developmentappears to be desirable, above all in light of the evolution of novelimaging methods in medical diagnostics.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and unless otherwise indicated, allparts and percentages are by weight.

The entire disclosures of all applications, patents and publications, ifany, cited above and below, and of corresponding application FederalRepublic of Germany P 39 38 992.8, filed Nov. 21, 1989, are herebyincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents Concentration-Time Curve of the cascade polymerdescribed in Example 8 as compared with "Magnevist" after intravenousadministration of 0.2 mmol/kg in rats. In spite of an identical dose, amarked difference is observed in the concentration of the two contrastmedia in the blood: "Magnevist" is distributed in the extracellularspace whereas the cascade polymer is distributed only in the vasal spaceand consequently reaches markedly higher concentrations.

FIG. 2 represents Coronary Projection Image of Head-Neck Region of aRat.

(a) Without contrast medium: the Signal intensity of the image is almostzero due to suppression of all tissues with relatively long T₁ times.

(b) One second after administration of 0.1 mmol Gd/kg of the titlecompound of Example 8: on account of intravasal distribution of thecontrast medium, only the T₁ time of the blood is extremely shortened sothat the signal intensity of the blood, in contrast to the surroundingtissue, is correspondingly high, and the result is an excellentcontrasting of the vessels.

(c) and (d) Four and, respectively 10 minutes after administration ofthe title compound of Example 8: in correspondence with the excretion ofthe compound, shortening of the T₁ time of the blood becomes lesspronounced whereby its signal intensity drops again.

FIG. 3 represents Coronary Projection Image of the Abdominal Region of aRat ten seconds after administration of the title compound of Example 8(0.25 mmol Gd/Kg). All of the significant blood vessels can clearly beobserved. A tumor is located in the zone of the left thigh, (on theright-hand side as seen by the observer), modifying the vascularstructure.

EXAMPLE 1

(a) 1,2-Epoxy-3-dibenzylaminopropane

At 0° C., 100 g (506.9 millimoles) of dibenzylamine (dissolved in 300 mlof methylene chloride) is added dropwise to a thoroughly stirredsuspension of 234.51 g (2.53 mol) of epichlorohydrin and 200 ml of 32%sodium hydroxide solution. The mixture is stirred for 2 hours at 0° C.and then 3 hours at room temperature. The mixture is diluted with 3 1 ofwater and extracted 3 times with 500 ml of methylene chloride. Theorganic phases are combined, dried over magnesium sulfate, andevaporated under vacuum. The remaining oil is flash-chromatographed onsilica gel (mobile phase: methylene chloride/hexane/acetone: 20/10/3).

Yield: 111.72 g of a colorless oil (87% of theory).

Analysis: C 80.60, H 7.56, N 5.53 (Calcd.), C 80.62, H 7.50, N 5.48(Found),

(b)10-(3-Dibenzylamino-2-hydroxypropyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

20 g ( 78.95 mmol) of the title compound of Example 1(a) and 20.51 g(59.21 mmol) of 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane (DO3A) are dissolved in a mixture of 50 ml of dioxane/200 ml of water,and the pH value is brought to 10 with 6N potassium hydroxide solution.The mixture is stirred for 24 hours at 40° C., evaporated to dryness,the residue taken up with 500 ml of water/500 ml of methanol, andextracted twice with 200 ml of tert-butylmethyl ether. The aqueoussolution is adjusted to pH 3 with 5N hydrochloric acid and evaporated todryness. The residue is concentrated under vacuum and then passed on toa column of poly-(4-vinylpyridine). The product is eluted with asolution of ethanol/water 1:1. After evaporation under vacuum, 22.37 g(63% of theory, based on DO3A) of the title compound is obtained as ahighly hygroscopic, vitreous solid (6.9% of water per analysis).

Analysis: C 62.08, H 7.56, N 11.68 (Calcd.), C 62.15, H 7.61, N 11.61(Found).

(c) Gadolinium Complex of10-(3-Dibenzylamino-2-hydroxypropyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

21 g (35.02 mmol) of the title compound of Example l(b) is dissolved ina solution of 150 ml of deionized water/50 ml of methanol, and 6.35 g(17.51 mmol) of gadolinium oxide is added thereto. The mixture isrefluxed for 2 hours, and 3 g of activated carbon is added. The solutionis filtered in the hot state, and the filtrate is evaporated to drynessunder vacuum.

Yield: 25.08 g (95% of theory) of a vitreous solid (5.2% water peranalysis).

Analysis:

C 49.39, H 5.61, N 9.29, Gd 20.86 (Calcd.),

C 49.41, H 5.70, N 9.25, Gd 20.88 (Found).

(d) Gadolinium Complex of10-(3-Amino-2-hydroxypropyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

24 g (31.83 mmol) of the title compound of Example l(c) is dissolved ina mixture of 250 ml of deionized water/150 ml of methanol, and 10 g ofpalladium catalyst (10% Pd on active carbon) is added. The mixture isthen hydrogenated for 24 hours at 50° C., filtered off from thecatalyst, and the filtrate is evaporated under vacuum.

Yield: 17.89 g (98% of theory) of the title compound as a vitreous solid(6.4% water per analysis).

Analysis: C 35.59, H 5.27, N 12.21) Gd 27.41 (Calcd.). C 35.51, H 5.34,N 12.16 Gd 27.36 (Found).

(e) Gadolinium Complex of10-(3-Isothiocyanato-2-hydroxypropyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

A solution of 4.81 g (41.83 mmol) of thiophosgene in 100 ml ofchloroform is added to a solution of 12 g (20.92 mmol) of the titlecompound of Example 1(d) in 500 ml of deionized water and 20 ml ofpolyvinylpyridine (Reillex). The two-phase solution is stirred for 10minutes at 40° C., then for one hour at room temperature, and filtered.The organic phase is separated and the aqueous phase extractedadditionally twice with 200 ml of chloroform- The aqueous phase is thenfreeze-dried.

Yield: 12.62 g (98% of theory) of a colorless powder (5.7% water peranalysis).

Analysis: C 35.11, H 4.58, N 11.3, S 5.21, Gd 25.54 (Calcd.), C 35.04, H4.64, N 11.31, S 5.15, Gd 25.48 (Found).

EXAMPLE 2

(a) 1-Dibenzylamino-5,6-epoxy-3-oxahexane

100 g (414 mmol) of N-dibenzylaminoethanol is dissolved in 200 ml ofmethylene chloride and added dropwise at 0° C. to a vigorously stirredmixture of 250 ml 50% sodium hydroxide solution, 7.03 g (20.7 mmol) oftetra-n-butylammonium bisulfate, 153.4 g (1.657 mol) of epichlorohydrin.The mixture is stirred for 8 hours at 0° C., overnight at roomtemperature, then diluted with 2 l of water and extracted three timeswith 500 ml of methylene chloride. The combined organic phases are driedover magnesium sulfate and evaporated under vacuum. The oil that remainsis subjected to flash chromatography (silica gel/mobile phase: methylenechloride/hexane/acetone 20/10/3).

Yield: 96.12 g (78% of theory) of a colorless oil.

Analysis: C 76.74, H 7.79, N 4.71 (Calcd.), C 76.68, H 7.85, N 4.66(Found).

(b)10-(6-Dibenzylamino-2-hydroxy-4-oxahexyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

34.34 g (115.47 mmol) of the title compound of Example 2(a) and 20 g(57.74 mmol) of 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane (DO3A) are dissolved in a mixture of 60 ml of dioxane/350 ml of water,and the pH is set at 10 with 6N potassium hydroxide solution. Themixture is stirred for 24 hours at 40° C. and worked up as described inExample l(b).

Yield: 26.39 g (71% of theory based on DO3A) of a vitreous solid (7.1%water per analysis).

Analysis: C 61.57, H 7.67, N 10.88 (Calcd.), C 61.49, H 7.80, N 10.79(Found).

(c) Gadolinium Complex of 10- (6-Dibenzylamino-2-hydroxy-4-oxahexyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

23 g (35.73 mmol) of the title compound of Example 2(b) is dissolved ina solution of 150 ml of deionized water/50 ml of methanol, and 6.48 g(17.86 mmol) of gadolinium oxide is added. The mixture is refluxed for 2hours, 3 g of active carbon is added, and refluxing is performed foranother hour. The solution is filtered in the hot state and the filtrateevaporated to dryness under vacuum, thus obtaining 27.65 g (97% oftheory) of the title compound as a vitreous solid (7.8% water peranalysis).

Analysis: C 49.67, H 5.81, N 8.78, Gd 19.71 (Calcd.). C 49.61, H 5.89, N8.71, Gd 19.61 (Found).

(d) Gadolinium Complex of 10-(6-Amino-2-hydroxy-4-oxahexyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

25 g (31.33 mmol) of the title compound of Example 2(c) is dissolved ina mixture of 250 ml of deionized water/150 ml of methanol, and 10 g ofpalladium catalyst (10% Pd on active carbon) is added. The mixture isthen hydrogenated for 24 hours at 50° C. The product is filtered offfrom the catalyst and the filtrate evaporated under vacuum.

Yield: 19.16 g (99% of theory) of the title compound as a vitreous solid(5.7% water per analysis)

Analysis:

C 36.94, H 5.55, N 11.34, Gd 25.45 (Calcd.),

C 36.88, H 5.59, , N 11.27, Gd 25.38 (Found).

(e) Gadolinium Complex of10-(6-Isothiocyanato-2-hydroxy-4-oxahexyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetrazacyclododecane

A solution of 5.58 g (48.56 mmol) of thiophosgene in 100 ml ofchloroform is added to a solution of 15 g (24.28 mmol) of the titlecompound of Example 2(d) in 500 ml of deionized water and 20 ml ofpolyvinylpyridine (Reillex). The two-phase solution is stirred for 10minutes at 40° C., then for one hour at room temperature, and filtered.The organic phase is separated and the aqueous phase extracted twicewith 200 ml of chloroform. Then the aqueous phase is freeze-dried.

Yield: 15.7 g (98% of theory) of a colorless powder (6.1% water peranalysis).

Analysis: C 36.4, H 4.89, N 10.6, Gd 23.83, S 4.86 (Calcd.) C 36.3, H4.9, N 10.51, Gd 23.7, S 4.78 (Found).

EXAMPLE 3 Gadolinium Complex of10-(9-Bromo-2-hydroxy-8-oxo-4-oxa-7-azanonyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

10 g (16.19 mmol) of the title compound of Example 2(e) is dissolved in50 ml of water and brought to pH 9 with 3N sodium hydroxide solution. At0° C., a solution of 4.25 g (21.04 mmol) of bromoacetyl bromide in 20 mlof dioxane is added dropwise thereto, and the pH is maintained at pH 9by addition of 3N sodium hydroxide solution. The mixture is stirred forone hour at 0° C., for two hours at room temperature, then evaporatedunder vacuum and the residue chromatographed ("Li-Chroprep RP-18"Merck/mobile phase: acetonitrile/H₂ O gradient). After evaporation ofthe main fractions under vacuum, 10.64 g (89% of theory) of the titlecompound is obtained as a crystalline solid (5.4% water per analysis).

Analysis: C 34.15, H 4.78, N 9.48, Gd 21.29, Br 10.82 (Calcd.), C 34.11,H 4.85, N 9.41, Gd 21.19, Br 10.75 (Found).

EXAMPLE 4

(a) 1-Dibenzylamino-5-hydroxy-3-oxapentane

A mixture of 50 g (475.56 mmol) of 2-(2-amino-ethoxy) ethanol and 144.6g (1.046 mol) of potassium carbonate in 600 ml of EtOH/60 ml of water isheated to 60° C. To this mixture is added dropwise within one hour178.95 g (1.046 mol) of benzyl bromide and then the mixture is refluxedfor 2 hours, evaporated under vacuum, the residue taken up with 1 literof methylene chloride, and filtered off from the salts. The filtrate isconcentrated under vacuum and purified by flash chromatography (silicagel/mobile phase: methylene chloride/hexane/acetone: 10/5/1).

Yield: 127.58 g (94% of theory) of a colorless oil.

Analysis: C 75.76, H 8.12, N 4.91 (Calcd.), C 75.71, H 8.18, N 4.85(Found).

(b) 1-Dibenzylamino-8,9-epoxy-3,6-dioxanonane

At 0° C., a solution of 125 g (438 mmol) of the title compound ofExample 4(a) in 200 ml of methylene chloride is added dropwise to athoroughly stirred suspension of 162.11 g (1.752 mol) ofepichlorohydrin, 8.2 g (24.15 mmol) of tetra-n-butylammonium bisulfate,and 250 ml of 50% sodium hydroxide solution. The mixture is stirred for8 hours at 0° C., overnight at room temperature. The mixture is dilutedwith 2 l of water and extracted twice with 500 ml of methylene chloride.The combined organic phases are dried over magnesium sulfate andevaporated under vacuum. The remaining oil is purified by flashchromatography (silica gel/mobile phase: methylenechloride/hexane/acetone: 20/10/3).

Yield: 116.5 g (78% of theory) of a colorless oil.

Analysis:

C 73.87, H 7.79, N 4.10 (Calcd.),

C 73.78, H 7.95, N 4.03 (Found).

(c)10-(9-Dibenzylamino-2-hydroxy-4,7-dioxanonyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

39.43 g (115.47 mmol) of the title compound of Example 4 (b) and 20 g(57.74 mmol) of 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane (DO3A) are dissolved in a mixture of 60 ml of dioxane/250 ml of water,and the pH is adjusted to 10 with 6N potassium hydroxide solution. Themixture is stirred for 24 hours at 40° C. and then worked up asdescribed in Example 1 (b).

Yield: 28.59 g (72% of theory based on DO3A) of a vitreous solid (6.3%water per analysis).

Analysis:

C 61.12, H 7.77, N 10.18 (Calcd.),

C 61.07, H 7.84, N 10.05 (Found).

(d) Gadolinium Complex of10-(9-Dibenzylamino-2-hydroxy-4,7-dioxanonyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

25 g (36.35 mmol) of the title compound of Example 4(c) is dissolved ina solution of 150 ml of deionized water/50 ml of methanol, and 6.59 g(18.17 mmol) of gadolinium oxide is added thereto. The mixture isrefluxed for 2 hours, 3 g of active carbon is added, and the mixture isrefluxed for another hour. The solution is filtered in the hot state andthe filtrate evaporated to dryness under vacuum.

Yield: 30.0 g (98% of theory) of the title compound as a vitreous solid(5.4% water per analysis).

Analysis: C 49.92, H 5.98, N 8.32, Gd 18.67 (Calcd.). C 49.83, H 5.90, N8.34, Gd 18.58 (Found).

Analogously, the corresponding europium complex is obtained with Eu ¹⁵¹Eu₂ O₃.

Analysis: C 50.24, H 6.02, N 8.37, Eu 18.16 (Calcd.), C 50.17, H 5.96, N8.29, Eu 18.09 (Found).

(e) Gadolinium Complex of10-(9-Amino-2-hydroxy-4,7-dioxanonyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

29 g (34.44 mmol) of the title compound of Example 4(d) is dissolved ina mixture of 250 ml of deionized water/150 ml methanol, and 10 g ofpalladium catalyst (10% Pd on active carbon) is added thereto. Then themixture is hydrogenated for 24 hours at 50° C., filtered off from thecatalyst, and the filtrate is evaporated under vacuum.

Yield: 22.56 g (99% of theory) of the title compound as a vitreous solid(6.5% water per analysis).

Analysis: C 38.11. H 5.79, N 10.58, Gd 23.76 (Calcd. C 38.05, H 5.8, N10.47 Gd 23.65 (Found).

(f) Gadolinium Complex of10-(9-Isothiocyanato-2-hydroxy-4,7-dioxanonyl)-l,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

A solution of 5.21 g (45.33 mmol) of thiophosgene in 100 ml ofchloroform is added to a solution of 15 g (22.66 mmol) of the titlecompound of Example 4(e) in 500 ml of deionized water and 20 ml ofpolyvinylpyridine (Reillex). The two-phase solution is stirred for 10minutes at 40° C., then for one hour at room temperature, and filtered.The organic phase is separated and the aqueous phase is extracted twicewith 200 ml of chloroform. Subsequently the aqueous phase isfreeze-dried.

Yield: 15.64 g (98% of theory) of a colorless powder (5.9% water peranalysis).

Analysis: C 37.54, H 5.15., N 9.95, Gd 22.34, S 4.55 (Calcd, C 37.49, H5.11, N 9.91, Gd 22.27, S 4.61 (Found).

EXAMPLE 5

(a)3,6,9-Tris(p-tolylsulfonyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene

At 100° C., a solution of 35.2 g (200 mol) of 2,6-bis(chloromethyl)pyridine (dissolved in 700 ml of dimethylformamide) isadded dropwise within 3 hours to 121.9 g (200 mmol) ofN,N',N"-tris(p-tolylsulfonyl)diethylenetriamine-N,N"-disodium salt in1600 ml of dimethylformamide. The mixture is agitated overnight at 100°C. Two liters of water is dripped into the hot solution, and the latteris allowed to cool down to 0° C. The precipitate is suctioned off andwashed with water. After drying under vacuum (60° C.), the product isrecrystallized from acetonitrile, thus obtaining 92.3 g (69% of theory)of the title compound as a colorless powder.

Analysis: C 57.46, H 5.43, N 8.38, S 14.38 (Calcd.), C 57.39, H 5.48, N8.35, S 14.35 (Found).

(b) 3,6,9,15-Tetraazabicyclo[9.3.1]pentadeca-1(15), 11,13-trieneTetrahydrosulfate

90.3 g (135 mmol) of the title compound of Example 5(a) is introducedinto 270 ml of concentrated sulfuric acid and stirred for 48 hours at100° C. The mixture is cooled to 0° C., and 1.35 l of absolute ether isadded dropwise thereto. The precipitate is suctioned off and extractedby stirring in 800 ml of methanol. After filtration and concentration,the product is dried under vacuum at 50° C.

Yield: 42.6 g (52.7% of theory) of a solid which deliquesces in the openair.

Analysis:

C 22.07, H 4.38, N 9.36, S 21.43 (Calcd.),

C 22.10, H 4.42, N 9.31, S 21.40 (Found).

(c) 3,6,9,15-Tetraazabicyclo[9.3-1]pentadeca-1(15),11,13-triene

40.0 g (66.8 mmol) of the title compound of Example 5(b) is dissolved in100 ml of water and adjusted to pH 11 with 32% strength sodium hydroxidesolution. The mixture is extracted 8 times with 150 ml of methylenechloride and dried over magnesium sulfate. After evaporation undervacuum, 9.79 g (71% of theory) of a yellowish powder is obtained.

Analysis: C 64.04, H 8.79, N 27.16 (Calcd.); C 63.91, H 8.85, N 26.98(Found).

(d)3,6,9-Tris(acetyl)-3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene

15.8 g (76.6 mmol) of the title compound of Example 5(c), 42.7 ml oftriethylamine (306.4 mmol) and 50 mg of dimethylaminopyridine (DMAP) aredissolved in 300 ml of absolute methylene chloride. The mixture iscombined with 28.9 ml (306.4 mmol) of acetic anhydride and stirredovernight at room temperature. The solvent is evaporated under vacuum,and the residue is taken up in 200 ml of 3% sodium carbonate solution.The mixture is extracted twice with 150 ml of methylene chloride. Afterdrying the organic phase over magnesium sulfate, the mixture isevaporated under vacuum. The residue is recrystallized from ether/ethylacetate, thus obtaining 23.93 g (94% of theory) of the title compound aswhite flakes.

Analysis:

C 61.42, H 7.28, N 16.86 (Calcd.),

C 61.48, H 7.37, N 16.80 (Found).

(e)3,6,9-Tris(acetyl)-3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-15-N-oxide

22.5 g (67.7 mmol) of the title compound of Example 5(d) is dissolved in100 ml of glacial acetic acid. To this solution is added 7.7 ml of a 30%strength hydrogen peroxide solution, and the mixture is heated for 4hours to 70° C. Then another 3.9 ml of 30% strength hydrogen peroxidesolution is added, and the mixture is stirred for another hour at 70° C.The mixture is then concentrated to one-third under vacuum, and gentlycombined with saturated sodium carbonate solution until an alkalinereaction is obtained. The mixture is extracted twice with 250 ml ofmethylene chloride and the organic phases are then dried over magnesiumsulfate. Evaporation under vacuum and crystallization from ether/ethylacetate yield 18.63 g (79% of theory) of the title compound as acrystalline powder.

Analysis:

C 58.60, H 6.94, N 16.08 (Calcd.),

C 58.47, H 6.88, N 16.14 (Found).

(f) 13-Nitro-3,6 , 9-tris (acetyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-15-N-oxide

17 g (48.8 mmol) of the title compound of Example 5(e) is dissolved in40 ml of 90% sulfuric acid and heated to 60° C. To this solution isadded dropwise 14 ml of concentrated nitric acid (d =1.36), and themixture is stirred for 3 hours at 60° C. The mixture is poured on ice,the precipitate is filtered and washed with a large amount of water.After drying under vacuum, an orange-colored powder is obtained which isrecrystallized from acetone.

Yield: 9.2 g (48% of theory) of yellow rhombi.

C 51.90, H 5.89, N 17.80 (Calcd.),

C 52.01, H 5.76, N 17.46 (Found),

(g) 13-Chloro-3,6 , 9-tris (acetyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-15-N-oxide

7.3 g (18.56 mmol) of the title compound of Example 5(f) is heated in 50ml of acetyl chloride for 4 hours to 50° C. The mixture is concentratedunder vacuum and the residue taken up in 200 ml of 3% strength sodiumcarbonate solution. The mixture is extracted three times with 100 ml ofchloroform and dried over magnesium sulfate. After removal of thesolvent under vacuum, the product is recrystallized from ether/ethylacetate.

Yield: 6.18 g (87% of theory) of a colorless crystalline powder.

Analysis: C 53.33, H 6.05, N 14.64, C19.26 (Calcd.) C 53.48, H 5.98, N14.71, C19.20 (Found).

(h)13-Chloro-3,6,9-tris(acetyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene

6.0 g (15.67 mmol) of the title compound of Example 5(g) is dissolved in300 ml of ethanol, 1 ml of concentrated hydrochloric acid is addedthereto, and the mixture is hydrogenated over Pd/C. After hydrogenabsorption has ceased, the mxiture is filtered off from the catalyst andevaporated under vacuum. The residue is taken up in 100 ml of 3%strength sodium carbonate solution and extracted twice with 100 ml ofchloroform. The organic phases are dried over magnesium sulfate andevaporated under vacuum. Crystallization of the residue from ether/ethylacetate yields 5.34 g (93% of theory) of the title compound as acolorless powder.

Analysis: C 55.66, H 6.32, N 15.27, C19.66 (Calcd.), C 55.57, H 6.38, N15.31, C19.59 (Found).

(i) 13-Chloro-3,6,9,15-tetraazabicyclo [9.3 .1]pentadeca-1(15),11,13-triene

5.1 g (13-9mmol) of the title compound of Example 5 (h) is dissolvedunder nitrogen in 50 ml of dioxane. To this mixture is added 6.24 g(55.6 mmol) of potassium tert-butylate, and the mixture is refluxedovernight, evaporated to dryness, taken up in 50 ml of water, andextracted 4 times with 100 ml of hot toluene. The combined toluenephases are dried over magnesium sulfate and evaporated under vacuum. Theresidue is purified by chromatography (silica gel/methanol/water/ammonia(aq. 33%) =10/1/1).

Yield: 3.01 g (90% of theory) of a slightly yellowish oil whichcrystallizes after a short time.

Analysis: C 54.88, H 7.12 , N 23.28, Cl 14.73 (Calcd.) C 54.93, H 7.06,N 23.41, Cl 14.81 (Found).

(k) 13-Chloro-3,6,9-tris(tert-butoxycarbonylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene

18.72 g (95.96 mmol) of bromoacetic acid tertbutyl ester is added to 7 g(29.08 mmol) of the title compound of Example 5 (i) and 10.17 g (95.96mmol) of sodium carbonate in 200 ml of acetonitrile, and the mixture isstirred at room temperature for 24 hours.

The mixture is evaporated under vacuum, the residue is taken up in 300ml of water and extracted three times with 200 ml of methylene chloride.After drying of the organic phases over magnesium sulfate, the mixtureis concentrated under vacuum and the remaining oil is chromatographed onsilica gel (mobile phase: methylene chloride/ethanol=15/1).

Yield: 14.08 g (83% of theory) of a colorless oil.

Analysis: C 59.73, H 8.12, N 9.61, Cl 6.08 (Calcd.) C 59.67, H 8.25, N9.58, Cl 6.01 (Found).

(1)13-Azido-3,6,9-tris(tert-butoxycarbonylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca1(15),11,13-triene

21 g (36.01 mmol) of the title compound of Example 5(k) is dissolved in200 ml of dimethylformamide, and 7.02 g (108 mmol) of sodium azide aswell as 951 mg (3.6 mmol) of 18-crown-6 are added thereto. The mixtureis stirred for 48 hours at 90° C. After cooling to room temperature, themixture is poured into 1.5 l of ice water and extracted three times with200 ml of ethyl acetate. After drying the organic phase over magnesiumsulfate, the mixture is evaporated and the remaining oil ischromatographed on silica gel (mobile phase: methylenechloride/ethanol=15/1).

Yield: 10.83 g (51% of theory) of a pale-yellow oil.

Analysis: C 59.06, H 8.03, N 16.63 (Calcd.), C 59.17, H 8.05, N 16.51(Found),

(m)13-Amino-3,6,9-tris(tert-butoxycarbonylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene

10 g (16.96 mmol) of the title compound of Example 5(1) is dissolved in400 ml of ethanol, and 1 g of Pearlman catalyst (20% palladium hydroxideon carbon) is added thereto. After 24 hours of hydrogenation undernormal pressure, the product is suctioned off from the catalyst andevaporated under vacuum. The remaining oil is chromatographed on silicagel (mobile phase: methylene chloride/methanol/triethyl-amine=10/1/0.05), thus obtaining 8.89 g (93% of theory) of a slightlyyellowish oil.

Analysis: C 61.78, H 8.76, N 12.42 (Calcd.), C 61.67, H 8.91, N 12.35(Found).

(n)13-Amino-3,6,9-tris(carboxymethyl)-3,6,9,15-tetraazabicycl[9.3.1]pentadeca-1(15),11,13-triene

8.2 g (14.55 mmol) of the title compound of Example 5(m) is dissolved in100 ml of trifluoroacetic acid and stirred for 6 hours at roomtemperature. After removal of the solvent by evaporation under vacuum,the residue is dissolved in 100 ml of water and passed over a columnfilled with poly(4-vinylpyridine). After evaporation under vacuum andcrystallization from methanol/acetone, 5.24 g (91% of theory) of astrongly hygroscopic solid is obtained.

Analysis: C 51.64, H 6.3, N 17.71 (Calcd.), C 51.74, H 6.3, N 17.63(Found).

(o) Gadolinium Complex of13-Amino-3,6,9-tris(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene

4.8 g (12.14 mmol) of the title compound of Example 5(n) is dissolved in35 ml of deionized water, and 2.2 g (6.07 mmol) of gadolinium oxide isadded. The mixture is stirred for 3 hours at 90° C., maintaining the pHby adding acetic acid at 5.5. The solution is filtered and passed over acolumn filled with poly(4-vinylpyridine). After treatment with activecarbon, the mixture is again filtered and freeze-dried.

Yield: 6.07 g (91% of theory) of an amorphous powder which, peranalysis, contains 12.1% water.

Analysis: C 37.15, H 4.06, N 12.74, Gd 28.61 (Calcd. C 37.08, H 4.17, N12.68, Gd 28.54 (Found).

(p) Gadolinium Complex of13-Isothiocyanato-3,6,9-tris(carboxymethyl)-3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene

5.49 g (10 mmol) of the title compound of Example 5(o) and 10 ml ofpolyvinylpyridine (Reillex) are dissolved in 100 ml of deionized water,and 3.45 g (30 mmol) of thiophosgene in 50 ml of chloroform is addedthereto. The mixture is stirred for 10 minutes at 40° C., then for onehour at room temperature, and filtered. The organic phase is separated,and the aqueous phase is extracted twice with 50 ml of chloroform. Thenthe product is freeze-dried.

Yield: 5.8 g (98% of theory) of a white powder (7.9% water peranalysis).

Analysis: C 36.54, H 3.41, N 11.84, Gd 26.58, S 5.43 (Calcd.), C 36.49,H 3.48, N 11.81, Gd 26.47, S 5.32 (Found).

EXAMPLE 6

(a) Hexamethyl Ester of the Tris (aminoethyl) amine Cascade Polymer

7.6 ml of tris(aminoethyl)amine (50 mmol) is dissolved in 10 ml ofmethanol and added dropwise to 54.5 ml (600 mmol) of methyl acrylate.The mixture is stirred for 3 days at room temperature and thenevaporated under vacuum. The remaining oil is precipitated frommethanol/ether/hexane.

Yield: 30.59 g (92.3% of theory) of a slightly yellowish oil.

Analysis: C 54.37, H 8.21, N 8.45 (Calcd.), C 54.32, H 8.29, N 8.43(Found),

(b) Hexaamine of the Tris(aminoethyl)amine Cascade Polymer

26.5 g of the hexamethyl ester described in Example 6(a) (40 mmol) isdissolved in 20 ml of methanol and gradually added dropwise to 242 ml ofethylenediamine (3.6 mol) and then stirred for 3 days at roomtemperature. The solution is evaporated under vacuum, and the remainingoil is reprecipitated from methanol/ether.

Yield: 31.25 g (94% of theory) of an oil having a slightly yellow color.

Analysis: C 52.03, H 9.46, N 26.96 (Calcd.), C 51.97, H 9.49, N 26.89(Found).

(c) Dodecamethyl Ester of the Tris (aminoethyl) amine Cascade Polymer

30.1 g (36.2 mmol) of the hexaamine described in Example 6(b) in 50 mlof methanol is added dropwise to 103 ml (1.14 mol) of methyl acrylate soslowly that the solution remains clear, and the latter is stirred for 5days at room temperature. After concentration under vacuum, the mixtureis repeatedly precipitated from methanol/ether/hexane.

Yield: 64.2 g (95.1%) of a yellowish oil.

The M+H⁺ peak is clearly recognizable in the FAB mass spectrum. Ananalytical sample showed the elementary analysis set forth below aftercorrection of methanol, determined by gas chromatography: C 54.12, H8.11, N 12.02 (Calcd.), C 54.01, H 8.19, N 11.98 (Found).

(d) Dodecaamine of the Tris(aminoethyl)amine Cascade Polymer

64.0 g (34.3 mmol) of the dodecamethyl ester described in Example 6 (c)is dissolved in 50 ml of methanol and gradually added dropwise to 870 mlof ethylenediamine (13 mmol) , and stirred for 5 days at roomtemperature. After concentration under vacuum, the mixture is repeatedlyprecipitated from methanol/ether until no ethylenediamine can bedetected any more by thin-layer chromatography.

Yield: 73.7 g (97%) of a viscous, yellowish oil.

The quasi molecule peak is clearly recognizable at 2201 in the FAB massspectrum. An analytical sample showed the following elementary analysisafter correction of methanol determined by gas chromatography: C 52.39,H 9.07, N 25.46 (Calcd.), C 52.29, H 9.21, N 25.71 (Found).

(e) 24-Methyl Ester of the Tris(aminoethyl)amine Cascade Polymer

68.0 g (30 mmol) of the 12-amine (Example 6d) is dissolved in 120 ml ofmethanol and added dropwise to 270 ml (3 mol) of methyl acrylate sogradually that the solution remains homogeneous (addition within 3hours). After 5 days, the mixture is worked up analogously to Example6(c).

Yield: 119.7 g (93.5%) of a yellowish oil.

The FAB mass spectrum shows the quasi molecule ion at m/e=4268. Ananalytical sample showed the following elementary analysis aftercorrection of methanol determined by gas chromatography: C 54.04, H8.08, N 13.13 (Calcd.), C 54.28, H 8.01, N 12.99 (Found).

(f) 24-Amine of the Tris (aminoethyl) amine Cascade Polymer

39.87 g (9.3 mmol) of the 24-methyl ester is dissolved in 100 ml ofmethanol and added dropwise to 1.5 1 of ethylenediamine (23 mol), andworked up after 7 days analogously to Example 6(d), thus obtaining 44.0g (95.9%) of a viscous yellow-colored oil. The compound is uniform inthe HPLC chromato-gram in 1-molar NaClO₄ in "Lichrospher" DIOL 100, 500,1000 (Merck).

Analysis: C 52.51, H 8.94, N 24.95 (Calcd.), C 52.17, H 8.72, N 25.27(Found).

(g)[10-Carboxy-3,6-bis(carboxymethyl)-9-ethoxycarbonylmethyl-3,6,9-triazadecanoyl]Derivativeof the 24-Amine of the Tris(aminoethyl)amine Cascade Polymer

4.94 g (1 mmol) of the aforedescribed 24-amine (Example 6f) is dissolvedin 300 ml of H₂ O. Within hours, 29.04 g (72 mmol) ofN3-(2,6-dioxomorpholinoethyl)-N⁶-(ethoxycarbonylmethyl)-3,6-diazaoctanedioic acid (Example 13a of EP0,331,616) is then added in portions in the solid form, the pH beingmaintained at 9.0 by adding 1N NaOH. The mixture is then stirred for 30minutes, adjusted to pH 7 with "Amberlite" IR 120 (H⁺ form), andsuctioned off from the ion exchanger. The solution is subjected toultra-filtration ("AMICON" YM5 membrane), and thereafter freeze-dried.

Yield: 13.6 g of a colorless, flaky powder. H₂ O content (Karl-Fischer):3.4%.

100 mg of the anhydrous complexing compound turn 24 mg Gd³⁺ into acomplex (indicator xylenol orange) (occupation value with DTPA>92%).

(h) Gd Complex of[10-Carboxy-3,6-bis(carboxymethyl)9-ethoxycarbonylmethyl-3,6,9-triazadecanoyl]Derivativeof the 24-Amine of the Tris(aminoethyl)amine Cascade Polymer

10.0 g of the complexing compound described in Example 6(g) is dissolvedin 500 ml of H₂ O and combined with 2.77 g of Gd₂ O₃ ( 2.40 g Gd),stirred for 30 minutes at 80° C., adjusted, after cooling, to pH 7 withion exchanger, membrane-filtered, and freeze-dried.

Yield: 12.1 g of a colorless, flaky lyophilized product.

H₂ O content: 5.6%,

Gd analysis (AAS): 17.9%,

T₁ relaxation (H₂ O): 12.98±0.27 [1/mmol·sec]; (plasma): 13.23±0.35[1/mmol·sec].

EXAMPLE 7

(a) 48-Methyl Ester of the Tris(aminoethyl)amine Cascade Polymer

19.8 g (4 mmol) of the 24-amine described in Example 6(f) is dissolvedin 100 ml of methanol and added dropwise within 5 hours to 200 ml (2.2mol) of methyl acrylate at 50° C., and stirred for 3 days at thistemperature. After repeated precipitation from methanol/ether/hexane,34.4 g (95%) of a viscous oil is obtained.

Analysis: C 54.01, H 8.07, N 13.59 (Calcd.), C 53.52, H 8.19, N 13.23(Found).

(b) 48-Amine of the Tris(aminoethyl)amine Cascade Polymer

23.2 g (2.5 mmol) of the 48-ester obtained in the preceding Example 7(a)is dissolved in 75 ml of methanol and added dropwise to 1000 ml ( 15mol) of ethylenediamine, and worked up after 7 days in analogy toExample 6(d) .

Yield: 25.0 g (96%) of a viscous oil.

The oil is uniform as per HPLC in 1-molar NaClO₄ on "Lichrospher"DIOL-100, 500, 1000 (Merck). Titration of an analytical sample with 1NHCl yields 96.4% of theory.

(c)[10-Carboxy-3,6-bis(carboxymethyl)-9-ethoxycarbonylmethyl-3,6,9-triazadecanoyl]Derivativeof the 48-Amine of the Tris(aminoethyl)amine Cascade Polymer

5.21 g (0.5 mmol) of the 48-amine described in Example 7(b) is dissolvedin 300 ml of H₂ O. Within 2 hours, 29.04 g (72 mmol) ofN3-(2,6-dioxomorpholinoethyl)-N⁶-(ethoxycarbonylmethyl)-3,6-diazaoctanedioic acid (Example 13a of EP0,331,616) is added to this solution in portions in the solid form. ThepH is maintained at 9.0 by the simultaneous addition of 1N NaOH. Themixture is worked up analogously to Example 6(g).

Yield: 13.3 g of a colorless lyophilized product.

H₂ O content (Karl-Fischer): 4.7%.

100 mg of the polymer complexes 24 mg of Gd³⁺.

(d) Gd Complex of the[10-Carboxy-3,6-bis(carboxymethyl)-9-ethoxycarbonylmethyl-3,6,9-triazadecanoyl]Derivativeof the 48-Amine of the Tris(aminoethyl)amine Cascade Polymer

10.0 g of the complexing compound described in Example 7(c) is dissolvedin 500 ml of H₂ O and combined with 2.77 g of Gd₂ O₃ 2.40 g Gd), stirredfor 30 minutes at 80° C., and worked up analogously to Example 6(h).

Yield: 12.2 g of a colorless flaky powder.

H₂ O content: 3.9%,

Gd analysis (AAS): 17.8% ,

T₁ relaxation (H₂ O): 13.52±0.37 [1/mmol·sec]; (plasma): 13.35±0.31[1/mmol·sec],

EXAMPLE 8 Gd Complex of the[10-Carboxy-3,6,9-tris(carboxymethyl)-3,6,9-triazadecanoyl]Derivative ofthe 48-Amine of the Tris(aminoethyl)amine Cascade Polymer

10.42 g (0.35 mmol) of the 48-DTPA ethyl ester disclosed in Example 7(c)is dissolved in 100 ml of 2N NaOH and stirred for 4 hours at roomtemperature. The alkaline solution is adjusted to pH 4 with "Amberlite"IR 120 (H⁺ form), suctioned off from the ion exchanger, and combinedwith 2.88 g of Gd₂ O₃ ( 2.50 g Gd), stirred for 30 minutes at 80° C.,adjusted to pH 7.2 with 1N NaOH, and the thus-produced solution issubjected to ultrafriltration ("AMICON" YM5 membrane). The desaltedsolution is finally freeze-dried, thus obtaining 12.1 g of a colorlesspowder.

H₂ O content (Karl-Fischer): 4.3%.

Gd analysis (AAS): 19.17%.

Melting point: 230° C. (onset of discoloration),

T₁ relaxation (H₂ O): 13.11±0.33 [1/mmol·sec]; (plasma): 13.09±0.27[1/mmol·sec],

Osmolality (0.5 tool/1 at 37° C.): 0.46 [osmol/kg],

Comparison "Magnevist": 1.96 [osmol/kg],

LD₅₀ (i.v. in mice): 30 mmol/kg.

Comparison "Magnevist":≦10 mmol/kg,

EXAMPLE 9

(a) Pentamethyl Ester of the Diethylenetriamine Cascade Polymer

5.54 ml of diethylenetriamine (50 mmol) is dissolved in 20 ml ofmethanol and added dropwise to 45.4 ml of methyl acrylate (500 mmol).The mixture is stirred for 5 days at room temperature and thenevaporated under vacuum. The remaining oil is reprecipitated frommethanol/ether/hexane.

Yield: 24.8 g (92.9%) of a slightly yellowish oil.

Analysis: C 54.02, H 8.12, N 7.87 (Calcd.), C 53.92, H 8.06, N 7.92(Found).

(b) Pentaamine of the Diethylenetriamine Cascade Polymer

21.3 g of the pentamethyl ester described Example 9 (a) (40 mmol) isdissolved in 20 ml of methanol and added dropwise slowly to 202 ml ofethylenediamine (3.0 mol) and then stirred for 3 days at roomtemperature. The solution is evaporated under vacuum and the remainingoil reprecipitated from methanol/ether.

Yield: 24.8 g (92%) of a slightly yellow-colored oil.

Analysis: C 51.69, H 9.42, N 27.02 (Calcd.), C 51.48. H 9.36, N 27.15(Found).

(c) Decamethyl Ester of the Diethylenetriamine Cascade Polymer

20.7 g of the pentaamine described in Example 9(b) in 35 ml of methanolis added to 68 ml (0.75 mol) of methyl acrylate so gradually that thesolution remains clear; the latter is stirred for 5 days at roomtemperature. After concentration under vacuum, the mixture is repeatedlyprecipitated from methanol/ether/hexane.

Yield: 39.8 g (84%) of a yellowish oil.

The M+H⁺ peak can be clearly recognized in the FAB mass spectrum. Aftercorrection of the methanol content determined by gas chromatography, ananalytical sample showed the following elementary analysis: C 54.00, H8.08, N 11.86(Calcd.), C 54.2, H 8.16, N 11.63 (Found).

(d) Decaamine of the Diethylenetriamine Cascade Polymer

39.5 g (25.7 mmol) of the decamethyl ester disclosed in Example 9 (c) isdissolved in 30 ml of methanol and added gradually to 520 ml (7.8 tool)of ethylenediamine, and stirred for 5 days at room temperature. Afterconcentration under vacuum, the mixture is repeatedly precipitated frommethanol/ether until no ethylenediamine can be detected any longer bythin-layer chromatography.

Yield: 44.9 g (96.2%) of a yellowish oil.

The quasi molecule peak can be readily seen in the FAB mass spectrum atm/e=1815. After correction of the methanol content determined by gaschromatography, an analytical sample showed the following elementaryanalysis: C 52.27, H 9.05, N 25.46 (Calcd.), C 52.11, H 9.09, N 25.67(Found).

(e) 20-Methyl Ester of the Diethylenetriamine Cascade Polymer

41.8 g (23 mmol) of the decaamine (Example 9d) is dissolved in 100 ml ofmethanol and added dropwise to 200 ml (2.2 tool) of methyl acrylate sogradually that the solution remains homogeneous (2 hours). After 5 days,the mixture is worked up analogously to Example 9 (c).

Yield: 72.0 g (88.5%) of a yellowish oil.

The FAB mass spectrum shows the quasi molecule at m/e=3536. Aftercorrection of the methanol content determined by gas chromatography, ananalytical sample showed the following elementary analysis: C 53.99, H8.06) N 13.07 (Calcd.), C 53.71, H 8.14, N 13.10 (Found).

(f) 20-Amine of the Diethylenetriamine Cascade Polymer

68.7 g (19.4 mmol) of the 20-methyl ester is dissolved in 100 ml ofmethanol and added dropwise to 1.5 1 (23 mol) of ethylenediamine, andworked up after 7 days analogously to Example 9 (d), thus obtaining 74.3g (93.5%) of a viscous oil. The oil is uniform in the HPLC in 1-molarNaClO₄ on "Lichrospher" DIOL-100, 500, 1000 (Merck). Titration of ananalytical sample with 1N HCl yields 97.8% of theory.

Analysis: C 52.46, H 8.93, N 24.95 (Calcd.), C 51.98, H 8.99, N 24.49(Found),

(g) 40-Methyl Ester of the Diethylenetriamine Cascade Polymer

20.5 g (5 mmol) of the 20-amine described in Example 9 (f) is dissolvedin 100 ml of methanol and added dropwise within 5 hours to 200 ml (2.2mol) of methyl acrylate at 50° C.; the mixture is stirred for 3 days atthis temperature. After repeated precipitation frommethanol/ether/hexane, 34.3 g (91%) of a viscous oil is obtained.

Analysis:

C 53.99, H 8.06, N 13.56 (Calcd.),

C 53.69, H 8.12, N 13.21 (Found),

(h) 40-Amine of the Diethylenetriamine Cascade Polymer

26.4 g (3.5 mmol) of the 40-ester obtained in the preceding Example 9(g)is dissolved in 100 ml of methanol and added dropwise to 1,170 ml (17.5mol) of ethylenediamine, and worked up after 7 days in analogy toExample 9 (d).

Yield: 28.7 g (94.6%) of a viscous, yellowish oil

The oil is uniform as per HPLC in 1-molar NaClO₄ on "Lichrospher"DIOL-100, 500, 1000 (Merck). Titration of an analytical sample with 1NHCl yields 95.3% of theory.

Analysis: C 52.54, H 8.88, N 24.73 (Calcd.), C 52.73, H 8.57, N 24.35(Found).

(i) Thioureido Conjugate of the Gd Complex of10-(6-Isothiocyanato-2-hydroxy-4-oxahexyl)1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecanewith the 40-Amine of the Diethylenetriamine Cascade Polymer

2.17 g (0.25 mmol) of the 40-amine described in Example 9 (h) isdissolved in 250 ml of H₂ O. Under nitrogen, 8.43 g (12 mmol, 1.2-foldexcess) of the isothiocyanate-Gd complex disclosed in Example 2(e) isadded in the solid form in portions to this mixture, and the latter isstirred overnight at room temperature. After ultrafiltration ("AMICON"YM-10 membrane), the conductivity of the solution is set at a minimum bymeans of ion exchanger ("Amberlite" IR 120, H⁺ form, and IRA 410, OH⁻form). The mixture is filtered off from the exchanger and freeze-dried.

Yield: 7.6 g (87%).

H₂ O content: 6.3%,

Gd analysis (AAS): 15.6%, T₁ relaxation (H₂ O): 12.43+0.51 [1/mmol·sec];(plasma): 13.19+0.42 [1/mmol- sec],

Analysis (anhydrous): C 40.39, H 5.87, N 14.10, Gd 17.94, S 3.66(Calcd.), C 40.67, H 6.15, N 13.88, Gd 16.88, S 3.47 (Found).

The following thioureido conjugates are obtained analogously:

from the isothiocyanate described in Example 1(e): C 39.65, H 5.70, N14.85, S 3.85, Gd 18.89 (Calcd.); C 40.12, H 5.55, N 14.31, S 3.39, Gd18.71 (Found).

from the isothiocyanate disclosed in Example 4 (f): C 41.07, H 6.03, N13.43, S 3.48, Gd 17.08 (Calcd.), C 40.69, H 5.97, N 13.57, S 3.61, Gd16.88 (Found).

as well as, from the isothiocyanate described in Example 5(p): C 40.83,H 4.87, N 15.29, S 3.97, Gd 19.45 (Calcd.), C 40.67, H 5.01, N 15.24, S3.70, Gd 19.11 (Found).

EXAMPLE 10

(a) Conjugate of tile 48-Amine, Partially Occupied by Sebacic AcidMonohydrazide, of the Tris(aminoethyl)amine Cascade Polymer withN3-(2,6-Dioxomorpholinoethyl)-N⁶-(ethoxycarbonylmethyl)-3,6-diazaoctanedioic Acid

0.48 g (1.5 mmol) of sebacic acid mono-(N-tert-butoxycarbonyl)hydrazide(Example 58b of EP 0,331,616) is dissolved in tetrahydrofuran andcombined, at -5° C., in succession with 4.16 ml (30 mmol) oftriethylamine and 0.15 ml (1.58 mmol) of ethyl chloroformate. After 15minutes, at -20° C., a solution of 6.51 g (30 mmol amino groups) of the48-amine described in Example 7(b) in tetrahydrofuran/H₂ O (10:1) isadded and the mixture is heated to room temperature. After 3 hours,tetrahydrofuran is distilled off, the mixture is diluted with H₂ O andcombined, at pH 9, in portions with 36.3 g (90 mmol) of N³-(2,6-dioxomorpholinoethyl)N⁶-(ethoxycarbonylmethyl)-3,6-diazaoctanedioic acid (Example 13a of EP0,331,616), and then set to pH 7 with dilute HCl. The solution isfiltered, the filtrate is purified by removing low-molecular componentsby way of an "AMICON" ultrafiltration membrane YM 10, and is finallyfreeze-dried. No impurities can be detected by thin-layercharomatography.

Yield: 16.2 g;

The thus-obtained polymeric boc-hydrazide is taken up without furtherpurification in trifluoroacetic acid, stirred for one hour at roomtemperature, and then precipitated with ether, suctioned off, and dried.The residue is set at pH 7.2 in H₂ O and freeze-dried.

Yield: 14.7 g.

Hydrazide content: 1.9 mol-%, H₂ O content: 8.3%, One gram of thiscompound complexes 192 mg of Gd³⁺.

(b) Gd Complex of the Conjugate of the 48-Amine, Partially Occupied bySebacic Acid Monohydrazide, of the Tris(aminoethyl)amine Cascade Polymerwith N³ -(2,6-Dioxomorpholinoethyl)-N⁶-(ethoxycarbonylmethyl)-3,6-diazaoctanedioic Acid

10.0 g of the complexing compound described in Example 10(a) isdissolved in 500 ml of H₂ O, combined with 2.21 g of Gd₂ O₃ ( 1.92 gGd³⁺), and stirred for one hour at 80° C. The resultant solution issubjected to ultrafiltration and then freeze-dried.

Yield: 11.7 g of a colorless powder.

Gd content (AAS): 15.8%.

Hydrazide content (by colorimetry): 1.8 mol-%.

Melting point: 258° C. (onset of discoloration).

T₁ relaxation (H₂ O): 12.23±0.41 [1/mmol·sec]; (plasma): 11.87±0.31[1/mmol·sec].

EXAMPLE 11 Gd Complex of the[10-Carboxy-3,6,9-tris(carboxymethyl)-3,6,9-triazadecanoyl]Derivative ofthe 24-Amine of the Tris(aminoethyl)amine Cascade Polymer

7.29 g (0.5 mmol) of the 24-DTPA-ethyl ester described in Example 6(g)is dissolved in 70 ml of 2N NaOH and stirred for 4 hours at roomtemperature. The alkaline solution is adjusted to pH 4 with "Amberlite"IR 120 (H⁺ form), suctioned off from the ion exchanger, and combinedwith 2.11 g of Gd₂ O₃ ( 1.83 g Gd³⁺), stirred for 30 minutes at 80° C.,adjusted to pH 7.2 with IN NaOH, and the thus-produced solution issubjected to ultrafiltration. The desalted solution is finallyfreeze-dried, thus obtaining 8.51 g (96.4%) of a colorless powder.

H₂ O content (Karl-Fischer): 3.9%,

Gd analysis (AAS): 19.84%,

Melting point: 250° C. (onset of discoloration),

T₁ relaxation (H₂ O): 11.17±0.48 [1/mmol·sec]; (plasma): 11.86±0.77[1/mmol·sec].

EXAMPLE 12

(a) 1,3-(N,N'-Tetrabenzyl) diamino-2-hydroxypropane

2.7 g (30 mmol) of 1,3-diamino-2-hydroxypropane and 14.3 ml (120 mmol)of benzyl bromide are refluxed with 8.3 g of potassium carbonate inethanol/H₂ O (10:1) overnight; then the suspension is evaporated todryness and taken up in water and toluene. The organic phase is driedover sodium sulfate and, after evaporation of the toluene under vacuum,the resulting oil is chromatographed on silica gel in ethylacetate/hexane (1:10).

Yield: 11.9 g (88%) of a colorless oil.

Analysis: C 82.63, H 7.60, N 6.22 (Calcd.), C 82.56, H 7.69, N 6.13(Found).

(b) 1,3-(N,N'-Tetrabenzyl)diamino-2-(oxiranylmethoxy)propane

9.01 g (20 mmol) of 1,3-(N,N'-tetrabenzyl) diamino-2-hydroxypropane(Example 12a) is dissolved in dichloromethane and added to a cooled (0°C.) solution of 4.69 ml (60 mmol) of epichlorohydrin and 340 mg oftetrabutylammonium bisulfate in 50% strength sodium hydroxide solutionand then the mixture is vigorously stirred overnight at 40° C. Thetwo-phase mixture is poured on about 100 ml of water, repeatedlyextracted with dichloromethane, and the combined organic phases aredried over MgSO₄. After evaporation of the solvent, an oil is obtained(9.83 g, 97%).

Analysis: C 80.60, H 7.56, N 5.53 (Calcd.), C 79.97, H 7.51, N 5.21(Found).

(c)6,6',6",6'",6"",6""'-Hexa[Bis[1-(N,N-dibenzylamino)-2-(N,N-dibenzylaminomethyl)-5-hydroxy-3-oxahexyl]amino]-6,6',6",6"',6"",6""'-hexadeoxy-α-cyclodextrin

12.58 g (10 mmol) of6,6',6",6"',6"",6""'-hexamino-6,6',6",6"',6"",6""'-hexadeoxy-α-cyclodextrinhexahydrochloride [J. Boger, R. J. Corcoran and J.-M. Lehn, Helv. Chim.Acta 61:2190-2218 (1978)]is combined in aqueous dioxane at pH 10 (setwith 1N sodium hydroxide solution) with 91.20 g (180 mmol, 1.5-foldexcess) of the epoxide described in Example 12(b), and stirred overnightat 50° C. The mixture is evaporated to dryness and chromatographed onsilica gel in dichloromethane/methanol (10:1).

Yield: 40.1 g (57%) of a pale-yellow oil.

Analysis: C 75.67, H 7.47, N 5.96 (Calcd.), C 75.19, H 7.59, N 5.39(Found).

(d)6,6',6",6"',6"",6""'-Hexa[bis(1-amino-2-aminomethyl-5-hydroxy-3-oxahexyl)amino]-6,6',6",6"',6"",6""'-hexadeoxy-α-cyclodextrin

35.24 g (5 mmol) of the benzyl-blocked 24-amine described in precedingExample 12(c) is suspended in aqueous ethanol and hydrogenated in anautoclave at 50° C. by means of H₂ /Pd (10 bar). The resultant solutionis evaporated to dryness and the thus-obtained amine is reacted withoutfurther purification.

Yield: 28.6 g (96%) of a pale-yellow oil.

An analytical sample was chromatographed on silica gel indioxane/water/concentrated ammonia (3:1:1) and showed the followinganalysis: C 72.48, H 7.60, N 7.04 (Calcd.), C 72.19, H 7.48, N 6.79(Found).

(e) Gd Complex of the Conjugate of N³ -(2,6-Dioxomorpholinoethyl)-N⁶-(ethoxycarbonylmethyl)-3,6-diazaoctanedioic Acid with6,6',6",6'",6"",6""'-Hexa[bis(1-amino-2-aminomethyl-5-hydroxy-3-oxahexyl)amino]-6,6',6",6"',6"",6""'-hexadeoxy-α-cyclodextrin

2.98 g (0.5 mmol) of the 24-amine disclosed in Example 7(d) is dissolvedin 150 ml of water. Then, within 2 hours, 14.52 g (36 mmol) of N³-(2,6-dioxomorpholinoethyl)-N⁶-(ethoxycarbonylmethyl)-3,6-diazaoctanedioic acid (Example 13a of EP0,331,616) is added in portions in the solid form, the pH beingmaintained at 9.5 by adding 1N NaOH. Subsequently, the ethyl ester issaponified by adding 25 ml of 32% strength sodium hydroxide solutionwithin 2 hours, set at pH 7 with "Amberlite" IR 120 (H⁺ form), and theion exchanger is suctioned off. The solution is subjected toultrafiltration ("AMICON" YM5) and freeze-dried- An analytical sampleshows that 100 mg of polymeric complexing compound absorb 24.2 mg of Gd(indicator: xylenol orange). The lyophilized product (8.40 g) isdissolved in 400 ml of water and combined with 2.3 g of Gd₂ O.sub. 3 (2.0 g Gd), stirred for 30 minutes at 80° C., set at neutral with ionexchanger, filtered, and freeze-dried.

Yield: 10.2 g of a colorless powder.

H₂ O content (Karl-Fischer): 4.8%,

Gd analysis (AAS): 17.0%.

Melting point: >250° C. (decomposition),

T₁ relaxation (H₂ O): 12.89±0.41 [1/mmol·sec]; (plasma): 13.17±0.32[1/mmol·sec].

EXAMPLE 13

(a)10-(2,6,7-Trihydroxy-4-oxaheptyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

19.56 g (103.92 mmol) of2,2-dimethyl-4-(2',3'-epoxy)propoxymethyl-1,3-dioxolane and 10 g (28.86mmol) of 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane (=DO3A)are dissolved in a mixture of 50 ml of dioxane/80 ml of water, and thepH value is set at 10 with 6N potassium hydroxide solution. The mixtureis stirred for 24 hours at 70° C., then evaporated to dryness, theresidue is taken up in 200 ml of water/50 ml of methanol, and extractedtwice with 100 ml of tert-butylmethyl ether. The aqueous solution isadjusted to pH 3 with 5N hydrochloric acid and evaporated to dryness.The residue is decocted (extracted) with 200 ml of methanol/80 ml ofdichloromethane. The mixture is cooled in an ice bath and filtered offfrom the precipitated potassium chloride. The filtrate is evaporatedunder vacuum, the residue is dissolved in 45 ml of water/20 ml ofethanol, and then passed over a column of poly(4-vinylpyridine). Theproduct is eluted with a solution of ethanol/water 1:3. Afterevaporation under vacuum, the residue is chromatographed on a reversedphase column (RP 18/mobile phase=gradient of water/tetrahydrofuran).After evaporation of the main fraction, 10.13 g (71% of theory) of astrongly hygroscopic, vitreous solid is obtained.

Analysis (based on anhydrous substance): C 48.57, H 7.74, N 11.33(Calcd.), C 48.46, H 7.81, N 11.24 (Found).

(b) Gd Complex of10-(2,6,7-Trihydroxy-4-oxaheptyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

8.56 g (17.3 mmol) of the title compound of Example 13(a) is dissolvedin 50 ml of deionized water, and 3.13 g (8.65 mmol) of gadolinium oxideis added. The mixture is heated to 90° C. for 3 hours. The cooledsolution is stirred for one hour with 3 ml of an acidic ion exchanger(AMB 252c) and 3 ml of a weakly alkaline ion exchanger (IRA 67). Theproduct is filtered off from the exchanger, and the filtrate isfreeze-dried.

Yield: 11.0 g (98% of theory) of a colorless amorphous powder.

Analysis (based on anhydrous substance): C 37.03, H 5.44, N 8.64; Gd24.24 (Calcd.), C 37.00, H 5.51, N 8.57, Gd 24.18 (Found),

(c) Gd Complex of the N-(5-Hydroxy-3-oxahexyl-DO3A)-48-amino CascadePolymer

38.93 g (60 mmol) of the Gd complex of Example 13(b) is dissolved in 400ml of methanol, combined with 25.67 g (120 mmol) of NaIO₄ and stirredfor 4 hours under exclusion of light. The product is then filtered offfrom the undissolved substance, and the filtrate is freeze-dried- Thelyophilized product is dissolved with 6.52 g (0.625 mmol=30 mmol NH₂) ofthe 48-cascade amine described in Example 7(b) in 750 ml of buffer, pH9.0 (Riedel de Haen, borax/HCl), and, after adding 11.32 g (180 mmol) ofsodium cyanoborohydride, stirred at room temperature for 6 days. Thesolution is then desalted via an "AMICON" ultrafiltration membrane YM5and finally freeze-dried.

Yield: 16.45 g (61% of theory),

H₂ O content (Karl-Fischer): 9.8%,

Gd determination (AAS): 15.75%,

T₁ relaxation (H₂ O): 12.35±0.14 [1/mmol·sec]; (plasma): 14.74±0.33[1/mmol·see].

EXAMPLE 14 Gd Complex of theN-(2-Carboxyethyl)-N-(5-hydroxy-3-oxahexyl-DO3A) -48-amino CascadePolymer

2.2 g of the polymer described in Example 13(c) with secondary amine inthe linkages between complex and backbone is dissolved in 25 ml ofmethanol and added dropwise to a mixture of 20 ml (220 mmol) of methylacrylate and 20 ml of methanol, and stirred for 3 days at roomtemperature. The solution is evaporated under vacuum, the pale-yellowoil is dissolved in 20 ml of 1N NaOH and saponified for 3 hours at roomtemperature. Then the product is neutralized with dilute HCl, and thesolution is desalted via an "AMICON" ultrafiltration membrane YM5 andfinally freeze-dried.

Yield: 2.20 g.

H₂ O content (Karl-Fischer): 7.7%.

Gd analysis (AAS): 14.93%.

T₁ relaxation (H₂ O): 11.47±0.14 [1/mol·sec]; (plasma): 13.38±0.07[1/mmol·sec].

Paper electrophoresis of the polymer at pH 9.0 (0.05-molar borax) and 10V/cm shows migration toward the anode whereas the starting compound(Example 13c) migrates to the cathode under the same conditions.

EXAMPLE 15 Gd Complex of theN-(1,2-Dicarboxyethyl)-N-(5-hydroxy-3-oxahexyl-DO3A)-48-amino CascadePolymer

Under ice cooling, 23 ml of triethylamine is added dropwise to 14.3 g(110 mmol) of maleic acid monomethyl ester (Tokyo Chemical Industry Co.Ltd.) in 15 ml of methanol. The mixture is allowed to reach roomtemperature, 2.20 g of the polymer described in Example 13(c) in 25 mlof methanol is added dropwise to this solution, and the mixture isagitated for 3 days at room temperature. Then the mixture is combinedwith diethyl ether, decanted from the separated oil, the remainingresidue is dissolved in 20 ml of 1N NaOH and saponified for 3 hours atroom temperature. Then the mixture is neutralized with dilute HCl andthe solution is desalted via an "AMICON" ultrafiltration membrane YM5and finally freeze-dried.

Yield: 2.13 g,

H₂ O content (Karl-Fischer): 7.9%,

Gd analysis (AAS): 14.53%.

T₁ relaxation (H₂ O): 11.93±0.27 [1/mmol·sec]; (plasma): 13.27±0.09[1/mmol·sec].

EXAMPLE 16 Gd Complex of theN-(Carboxymethyl)-N-(5-hydroxy-3-oxahexyl-DO3A)-48-amino Cascade Polymer

2.20 g of the polymer disclosed in Example 13(c) is dissolved in 25 mlof H₂ O and set at pH 10 by adding 1N NaOH. A solution of 2.6 g (22mmol) of sodium chloroacetate in 20 ml of H₂ O is added slowly dropwiseat 50° C. to this solution, and the pH is maintained at 10 by additionof 1N NaOH. After the addition step is completed, the mixture is stirredovernight at this temperature, then neutralized with dilute hydrochloricacid, and the solution is desalted via an "AMICON" ultrafiltrationmembrane YM5. After freeze-drying, 2.3 g of a flaky pwoder is obtained.

H₂ O content (Karl-Fischer): 10.5%,

Gd analysis (AAS): 15.12% .

T₁ relaxation (H₂ O): 12.25±0.37 [1/mmol·sec]; (plasma):12.93±0.14[1/mmol·sec].

EXAMPLE 17 Gd Complex of theN-(Carboxymethoxyacetyl)-N-(5-hydroxy-3-oxahexyl-DO3A)-48-amino CascadePolymer

2.20 g of the polymer described in Example 13 (c) is dissolved in 25 mlof H₂ O and set to pH 9 by adding 1N NaOH. Under agitation, 850 mg (6.6mmol) of diglycolic acid anhydride (Fluka) is added to this solution inportions, the pH being maintained at 9 by adding 2N NaOH. After theaddition step is completed, the mixture is stirred for 15 minutes,neutralized with dilute hydrochloric acid, subjected to ultrafiltration("AMICON" YM5), and finally freeze-dried.

Yield: 2.43 g,

H₂ O content (Karl-Fischer): 8.3%,

Gd analysis (AAS): 14.82%,

T₁ relaxation (H₂ O): 11.45±0.23 [1/mmol·sec]; (plasma): 13.74±0.20[1/mmol·sec].

EXAMPLE 18 Thioureido Conjugate of the Gd Complex of10-(6-Isothiocyanato-2-hydroxy-4-oxahexyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecanewith the Gd Complex of the N-(5-Hydroxy-3-oxahexyl-DO3A)-48-aminoCascade Polymer

2.20 g of the polymer described in Example 13(c) is dissolved in 25 mlof H₂ O. Under nitrogen, 3.09 g (4.7 mmol) of the isothiocyanate-Gdcomplex disclosed in Example 2(e) is added in portions in the solid formto this solution, and the mixture is stirred overnight at roomtemperature. After ultra-filtration ("AMICON" YM-10 membrane), theconductivity of the solution is set at a minimum by means of an ionexchanger ("Amberlite" IR 120, H⁺ form and IRA 410, OH⁻ form). Theproduct is filtered off from the exchanger and freeze-dried.

Yield: 3.31 g,

H₂ O content (Karl-Fischer): 7.3%,

Gd analysis (AAS): 15.32%,

T₁ relaxation (H₂ O): 12.79±0.30 [1/mmol·sec]; (plasma): 14.21±0.05[1/mmol·sec].

EXAMPLE 19

(a)10-(2,3,4-Trihydroxybutyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

10.0 g (28.87 mmol) of1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane (DO3A) isdissolved in 40 ml of water, and the pH is set at 13 with 5N sodiumhydroxide solution. A solution of 6.24 g (43.30 mmol) of2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethylene oxide (DE 3,150,917) in 10 mlof dioxane is added thereto, and the mixture is stirred for 24 hours atroom temperature. The mixture is diluted with 60 ml of water andextracted three times with 50 ml of ether. The aqueous phase is broughtto pH 2 with 10% strength hydrochloric acid and evaporated. The residueis dissolved in a small amount of water and passed to a cation exchangecolumn (IR 120). After flushing with water, the ligand is eluted with0.5-normal aqueous ammonia solution. The fractions are evaporated, theammonium salt is taken up in a small amount of water and passed over ananion exchange column (IRA 67). The mixture is first washed with waterand then eluted with 0.5-normal aqueous formic acid. The product isevaporated under vacuum, the residue is dissolved in a small amount ofhot methanol, and acetone is added, thus crystallizing the titlecompound.

Yield: 11.31 g (87% of theory) of a white hygroscopic powder.

H₂ O content (Karl-Fischer): 11.1%,

Analysis (based on anhydrous substance): C 47.99 H 7.61 N 12.44(Calcd.), C 47.93 H 7.67 N 12.40 (Found).

(b) Gadolinium Complex of10-(2,3,4-Trihydroxybutyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

10.0 g (22.2 mmol) of the compound obtained according to Example 19 (a)is dissolved in 60 ml of deionized water, and 4.02 g (11.1 mmol) ofgadolinium oxide is added. The mixture is heated for 3 hours to 90° C.The cooled solution is stirred with respectively 2 ml of acidic ionexchanger (IR 120) and 2 ml of alkaline exchanger (IRA 410) for one hourat room temperature, filtered off from the exchanger, and the filtrateis briefly boiled with active carbon. After filtration andfreeze-drying, a white, amorphous powder is obtained.

Yield: 12.76 g (95% of theory).

H₂ O content (Karl-Fischer): 12.3%,

Analysis (based on anhydrous substance): C 35.73, H 5.17, Gd 25.99, N9.26 (Calcd.), C 35.68, H 5.24, Gd 25.931 N 9.21 (Found).

(c) Gd Complex of the N-(2-Hydroxypropyl-DO3A)-48-amino Cascade Polymer

13.8 g (20 mmol) of the Gd complex of Example 19 (b) is dissolved in 120ml of methanol, combined with 8.56 g (40 mmol) of NaIO₄ , and stirredfor 4 hours under exclusion of light. Then the mixtrure is filtered offfrom the undissolved matter, and the filtrate is freeze-dried. Thelyophilized product is dissolved with 2.17 g (0.208 mmol=10 mmol --NH₂)of the 48-cascade amine described in Example 7 (b) in 250 ml of buffer,pH 9.0 (Riedel de Haen, borax/HCl), and, after addition of 3.77 g (60mmol) of sodium cyanoborohydride, stirred for 6 days at roomtemperature. The solution is then desalted via an "AMICON"ultrafiltration membrane YM5 and finally freeze-dried.

Yield: 5.87 g,

H₂ O content (Karl-Fischer): 8.9%.

Gd analysis (AAS): 15.93%,

T₁ relaxation (H₂ O): 13.22±0.23 [1/mmol·sec]; (plasma): 14.39±0.12[1/mmol·sec].

EXAMPLE 20 Gd Complex of theN-(carboxymethoxyacetyl)-N-(2-hydroxypropyl-DO3A)-48-amino CascadePolymer

1.7 g of the polymer described in Example 19(c) is dissolved in 20 ml ofH₂ O and set to pH 9 by addition of 1N NaOH. Under agitation, 772 mg (6mmol) of diglycolic acid anhydride (Fluka) is added thereto in portions,the pH being maintained at 9 by addition of 2N NaOH. After the additionis finished, the mixture is further stirred for 15 minutes, neutralizedwith dilute hydrochloric acid, subjected to ultrafiltration ("AMICON"YM5), and finally freeze-dried.

Yield: 1.90 g,

H₂ O content (Karl-Fischer): 10.7%,

Gd analysis (AAS): 14.93%,

T₁ relaxation (H₂ O): 13.52±0.22 [1/mmol·sec]; (plasma): 15.01±0.37[1/mmol·sec].

EXAMPLE 21 Conjugate of the Gd Complex of10-(9-Bromo-2-hydroxy-8-oxo-4-oxa-7-azanonyl)-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecanewith the Gd Complex of the N-(5-Hydroxy-3-oxahexyl-DO3A)-48-aminoCascade Polymer

1.7 g of the polymer disclosed in Example 19 (c) is dissolved in 20 mlof H₂ O and set to pH 9.5 by addition of 2N NaOH. At 40° C., 4.43 g (6mmol) of the Gd complex described in Example 3 is added thereto underagitation, the pH being maintained at 9.5 by adding 2N NaOH. After 24hours at 40° C., the mixture is neutralized with dilute hydrochloricacid, subjected to ultra filtration ("AMICON" YM5), and finallyfreeze-dried.

Yield: 2.45 g,

H₂ O content (Karl-Fischer): 9.7%,

Gd analysis (AAS): 15.72%,

T₁ relaxation (H₂ O): 13.07±0.23 [1/mmol·sec]; (plasma): 14.39±0.15[1/mmol·sec].

EXAMPLE 22 Yttrium-90 complex of the[10-Carboxy-3,6,9-tris-(carboxymethyl)-3,6,9-triazadecanoyl]Derivativeof the 48-Amine of the Tris (aminoethyl) amine Cascade Polymer

1.04 g (35 μmol) of the 48-DTPA-ethyl ester described in Example 7(c) isdissolved, as described in Example 8, in 10 ml of NaOH, stirred for 4hours at room temperature, and set to pH 7 with "Amberlite" IR 120 (H⁺form). The mixture is suctioned off from the ion exchanger and thesolution is freeze-dried, thus obtaining 0.98 g. Of this amount, 9.8 mgis added to yttrium-90 (yttrium chloride, Amersham; 11 μCi) in 100 μl of0.1-molar tetramethylammonium acetate, pH 5. After 10 minutes, controlby thin-layer chromatography reveals that complexing has taken placecompletely. The mixture is subsequently dialyzed via a "Centricon" 10ultrafiltration unit (Amersham).

EXAMPLE FOR IN VIVO NMR DIAGNOSTICS

The test animals (rats, Wistar Han ) are anesthetized for the nuclearspin tomograph examination ("Rompun" + "Ketavet") and are provided witha catheter in the caudal vein for administration of the contrast medium.The test is performed in an MRI experimental device by General Electric(field strength 2 tesla). The images are produced with a saturationinversion projection (SIP) sequence. This is a standard saturation andinversion recovery pulse sequence wherein the signals of all tissuesexcept for the blood are suppressed. Prior to utilization of thecontrast medium, the sequence is optimized to minimum intensity [typicalvalues: T (saturation)=50-60 msec; T (inversion)=40-50 msec].

FIG. 2 shows the angiographic visualization of the head-neck region of arat. The scan without contrast medium (top left-hand side) shows almostno signal at all. (Imaging period for all scans is 1 minute.) Afteradministration of the title compound of Example 8 (0.1 mmol Gd/kg), anexcellent contrasting of the vessels results, becoming weaker with timein correspondence with the elimination of the compound (1b=1 second,1c=4 minutes, and 1d=10 minutes p.i.).

FIG. 3 shows an angiographic scan of the abdominal zone of a rat (LewMol O) produced after a dose of 0.25 mmol Gd/kg under otherwiseidentical conditions as in the preceding example. The animal carries atumor at the left thigh (on the right-hand side as seen by theobserver). The vessel structures, altered in this area, and/or thevessels feeding the tumor, as well as many other relevant vessels of theabdominal zone, are excellently contrasted.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A cascade polymer, being either a chelating agentcapable of being chelated with at least five ions of an element ofatomic numbers 21-29, 39, 42, 44 or 57-83 or a chelate --K, with atleast give of said ions, of formula I ##STR15## wherein A is anitrogen-containing cascade nucleus of basis multiplicity b of theformula: ##STR16## wherein R², R³ and R⁴ are, in each case independentlyof one another, a covalent bond or

    --(CH.sub.2).sub.k --(C.sub.6 H.sub.4).sub.r --(CH.sub.2).sub.1 --N<

g is the number 2, 3, 4 or 5, t is the number 1, 2, 3, 4, 5, 6, 7 or 8,l is the number 0, 1, 2, 3, 4 or 5, r is the number 0 or 1, n is thenumber 0, 1, 2, 3 or 4, m is the number 0, 1, 2, 3 or 4, k is the number1, 2, 3, 4 or 5, a is the number 2, 3, 4 or 5, W is CH, CH₂, NH or anitrogen atom, C₁ is (CH₂)_(k) --N<, C₂, C₃, C₄ and C₅ are, in each caseindependently, a hydrogen atom or (CH₂)_(f) --N<, f is the number 1, 2,3, 4 or 5, j is the number 6, 7 or 8, Y¹ and Y² are, in each caseindependently of each other, a hydrogen atom, CH₂ --CH(OH)--CH₂ N< or(CH₂)_(a) --N<, and Y³ is a nitrogen atom, O--CH₂ --CH(OH)--CH₂ N< orO--(CH₂)₉ --N<, a and g are as defined above, means a single or doublebond, with the proviso that, if Y³ is a nitrogen atom, Y¹ and Y² arehydrogen; S is a repeating unit of the formula: ##STR17## a is 2, 3, 4or 5, α and β each independently is a hydrogen atom or (CH₂)_(o), `γmeans (CH₂)_(f), each f independently is the number 1, 2, 3, 4 or 5, andr is the number 0 or 1, and k is 1, 2, 3, 4 or 5, l is 0, 1, 2, 3, 4 or5, o is 0, 1, 2, 3, 4 or 5, with the proviso that l and o are not bothzero at the same time; N is a nitrogen atom, Z¹ and Z², for the first topenultimate generation, in each case are ##STR18## and, for the lastgeneration, Z¹ is hydrogen, a C₁ -C₁₀ -alkyl, C₂ -C₁₀ -carboxylic acylor C₁ -C₁₀ -alkylsulfonyl, each optionally containing 1-3 carboxy, 1-3sulfonic acid, 1-5 hydroxy groups and/or 1-3 oxa atoms, or is theresidue of a chelating agent or chelate K, and Z² is, to an extent of96-100% of the total Z² content, the residue of an attached chelatingagent or chelate k and, to an extent of 4-0%, V', wherein V' is V havingat its end a functional group or, linked via this functional group,which is: --NH₂ ; --NHR; --NHNH₂ ; NRNH₂ ; SH; OH; --COCH₂ ; CH═CH--CO₂R; --NCS; NCO; ##STR19## wherein R and R' are identical or different andin each case are hydrogen, a saturated or unsaturated C₁ -C₂₀-hydrocarbyl residue optionally substituted by a phenyl group, or aphenyl group, or, linked via this function group, a bio- ormacromolecule, V being a straight-chain, C₁ -C₂₀ -alkylene group whichoptionally contains imino, phenylene, phenylenoxy, phenylenimino, amide,hydrazide, uredio, thioureido, carbonyl and/or ester group(s) and/oroxa, sulfur and/or nitrogen atom(s), and is optionally substituted bycarboxyl carboxylalkyl, hydroxy, mercapto, imino, epoxy, oxo, thioxo,and/or amino, b is a number 1 through 50, and s is a number 1 to 3,wherein the reproduction units S need be identical only for a givengeneration, and the residue of the chelating agent or chelate is aresidue of Formulae I A, I B or I C; ##STR20## bound to the terminalnitrogen atoms of the final generation by a --CH₂ CO-- or V group, n andm in each case independently is 0, 1, 2, 3 or 4, n and m adding up to nomore than 4, k is the number 1, 2, 3, 4 or 5, l is the number 0, 1, 2,3, 4 or 5, q is the number 0, 1 or 2, U is CH₂ X or V, X is in each caseindependently --COOH or V' wherein, if the molecule contains V', atleast 0.1% of the substituents X stand for V', B, D and E, beingidentical or different, are in each case --(CH₂)_(a) with a being thenumber 2, 3, 4 or 5, R¹ is V or a hydrogen atom, with the proviso thatR¹ is V only if U is CH₂ X at the same time, and that U is V only if R¹is a hydrogen atom at the same time, and wherein optionally, a portionof the COOH groups are present in the form of a corresponding ester of aC₁ -C₆ -alkyl group or amide of a saturated or unsaturated, linear,branched or cyclic hydrocarbon of 1-5 carbon atoms, optionallysubstituted with 1 to 3 hydroxy or C₁ -C₄ -alkyoxy or a 5- or 6-memberedring including the amide nitrogen; or a salt thereof with a cation of anin organic and/or organic base, an amino acid or an amino acid amide. 2.A cascade polymer according to claim 1, containing at least five ions ofan element of atomic numbers 21-29, 39, 42, 44 or 57-83 bound to achelating agent K.
 3. A cascade polymer according to claim 1, whereinthe bio- or macromolecule(s) optionally contained in V' is/are anantibody or antibodies or antibody fragment of fragments.
 4. A cascadepolymer according to claim 1, wherein the bio- or macromolecule(s)optionally contained in V' is/are a protein or proteins.
 5. A cascadepolymer of claim 4, wherein the protein is albumin, globulin or lectin.6. A cascade polymer according to claim 1, wherein the bio- ormacromolecule(s) optionally contained in V' is/are a polysaccharide orpolysaccharides.
 7. A cascade polymer of claim 6, wherein thepolysaccharide is starch, dextran or dextrin.
 8. A cascade polymeraccording to claim 1, wherein V is ##STR21##
 9. A cascade polymeraccording to claim 1 which has 2-6 generations of reproduction units.10. A pharmaceutical agent comprising at least one cascade polymeraccording to claim 1 and a pharmaceutically acceptable carrier.
 11. Apharmaceutical agent comprising at least one cascade polymer accordingto claim 2 and a pharmaceutically acceptable carrier.